Retinoid replacements and opsin agonists and methods for the use thereof

ABSTRACT

Compositions of and methods for using synthetic retinoids as retinoid replacements and opsin agonists are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a U.S. National Phase Application under 35 U.S.C. §371 ofInternational Application No. PCT/US2004/007937, filed Mar. 15, 2004,which claims the benefit of U.S. Provisional Patent Application No.60/455,182, filed Mar. 14, 2003, the disclosure of which is incorporatedby reference herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This research was supported by United States Public Health ServiceGrants EY01730, EY02048, EY08061, EY09339, EY11850, EY13385 and EY66388from the NEI, National Institutes of Health grants. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

A diminished visual acuity or total loss of vision may result from anumber of eye diseases or disorders caused by dysfunction of tissues orstructures in the anterior region of the eye and/or posterior region ofthe eye. The eye is divided anatomically into an anterior and posteriorsegment. The anterior segment includes the come a, anterior chamber,iris and ciliary body (anterior choroid), posterior chamber andcrystalline lens. The posterior segment includes the retina with opticnerve, choroid (posterior choroid) and vitreous. The posterior portionof the eyeball supports the retina, choroid and associated tissues.

Examples of eye disorders resulting from the pathologic conditions ofstructures in the anterior segment of the eye are dry eye syndrome,keratitis or corneal dystrophy, cataracts, and glaucoma. Disease ordisorders of the posterior segment of the eye in general are retinal orchoroidal vascular diseases or hereditary diseases such as LeberCongenital Amaurosis. Age related macular degeneration (AMD) is one ofthe specific diseases associated with the posterior portion of theeyeball and is the leading cause of blindness among older people. AMDresults in damage to the macula, a small circular area in the center ofthe retina. Because the macula is the area which enables one to discernsmall details and to read or drive, its deterioration may bring aboutdiminished visual acuity and even and to read or drive, itsdeterioration may bring about diminished visual acuity and evenblindness. The retina contains two forms of light receiving cells, rodsand cones, that change light into electrical signals. The brain thenconverts these signals into the images. The macula is rich in conecells, which provides central vision. People with AMD sufferdeterioration of central vision but usually retain peripheral sight.

There are several types of AMD. The “dry” (non-exudative) type accountsfor about 90% of AMD cases. The “wet” (exudative) form afflicts onlyabout 10% of AMD patients. However, the wet form is a more seriousdisease than the dry form and is responsible for about 90% of theinstances of profound visual loss resulting from the disease. Wet AMDoften starts abruptly with the development of tiny, abnormal, leakyblood vessels termed CNVs (chorodial new vessels), directly under themacula. In most patients, this leads to scarring and severe centralvision loss, including distortion, blind spots, and functionalblindness.

Signs of AMD such as drusen, which are abnormal yellow deposits underthe retina, can be present even in patient with normal vision. Drusenlook like specks of yellowish material under the retina. They aredeposits of extracellular material that accumulate between retinalpigment epithelium (RPE) and Bruch's Membrane. The RPE is a specializedcell layer that ingests used-up outer tips of the rod and cone cells andprovides them with essential nutrients (e.g., vitamin A derivatives).Bruch's membrane is a noncellular structure (composed mostly ofcollagen) that separates the RPE from the choroidal circulation below.The choroidal circulation provides blood supply to the rods, cones andRPE cells. A few small drusen normally form in the human eye, usuallyafter age 40. AMD, in contrast, is almost always associated with abuild-up of additional drusen. Drusen occur in two forms. Hard drusenare small, solid deposits that apparently do no harm when present insmall numbers. Soft drusen are larger and may have indistinct borders.As soft drusen build up between the RPE and Bruch's membrane, they liftup the RPE and force the two layers apart.

Drusen develop long before the abnormal vessels of wet AMD. Threecharacteristics of soft drusen are risk factors for developing CNV: Thepresence of five or more drusen deposits; drusen size greater than 63micrometers (about the thickness of a human hair); and the clumping ofthe drusen deposits. Some evidence suggests soft drusen are instrumentalin the spread of abnormal vessels, but whether they stimulate vesselgrowth (angiogenesis) or simply provide space for them by lifting up theRPE remains unclear.

Two networks of blood vessels nourish the retina, one located on theretinal surface and the other located deep in the retina, external toBruch's membrane. The abnormal vessels of AMD originate in the lowernetwork of vessels, called the choroidal circulation. These vessels maketheir way through Bruch's membrane and spread out under the RPE. Bloodand fluids leak from them and cause the photoreceptor cells todegenerate and the macula to detach from the cells under it.

Slightly blurred or distorted vision is the most common early symptom ofAMD. Visual loss with dry AMD usually progresses slowly while visualloss with wet AMD proceeds more rapidly and may occur over days orweeks. Patients who have wet AMD in one eye are at increased risk ofdeveloping CNVs in the other eye. The magnitude of the risk varies,depending on the appearance of the second eye. The risk is greater ineyes with numerous large drusen, with abnormal pigment changes in themacula, and in patients with a history of high blood pressure.

AMD is now the leading cause of legal blindness in the western world.Reactions that go on in the RPE lead to oxidative products that in turnlead to cell death and neovascularization. This excess metabolism leadsto the formation of drusen under the RPE.

Other eye diseases also affect photoreceptor function in the eye.Retinitis Pigmentosa represents disease caused by defects in manydifferent genes. They all have a final common pathway of night blindnessand peripheral vision loss that can lead to narrowing of the visualfield and eventual loss of all vision in many patients. The rodphotoreceptors are usually primarily affected and most of the genedefects leading to the disease occur in genes that are expressedpredominantly or only in the rod cells.

One autosomal dominant form of Retinitis Pigmentosa comprises an aminoacid substitution in opsin, a proline to histidine substitution at aminoacid 23. This defect compromises 10-20% of all Retinitis Pigmentosacases. This abnormal opsin protein forms a protein aggregate thateventually leads to cell death.

Leber Congenital Amaurosis is a very rare childhood condition thataffects children from birth or shortly there after. It affects both rodsand cones. There are a few different gene defects that have beenassociated with the disease. These include the genes encoding the RP65and LRAT proteins. Both result in a person's inability to make11-cis-retinal in adequate quantities. In the RP65 defectiveindividuals, retinyl esters build up in the RPE. LRAT-defectiveindividuals are unable to make esters and subsequently secrete anyexcess retinoids.

Retinitis Punctata Albesciens is another form of Retinitis Pigmentosathat exhibits a shortage of 11-cis-retinal in the rods. Aging also leadsto the decrease in night vision and loss of contrast sensitivity due toa shorting of 1-cis-retinal. Excess unbound opsin is believed torandomly excite the visual transduction system. This can create noise inthe system, and thus more light and more contrast is necessary to seewell.

Congenital Stationary Night Blindness (CSNB) and Fundus Albipunctatusare a group of diseases that are manifested as night blindness, butthere is not a progressive loss of vision as in the RetinitisPigmentosa. Some forms of CSNB are due to a delay in the recycling of11-cis-retinal. Fundus Albipunctatus until recently was thought to be aspecial case of CSNB where the retinal appearance is abnormal withhundreds of small white dots appearing in the retina. It has been shownrecently that this is also a progressive disease although much slowerthan Retinitis Pigmentosa. It is caused by a gene defect that leads to adelay in the cycling of 11-cis-retinal.

Currently, there are few treatments for retinoid deficiency. Onetreatment, a combination of antioxidant vitamins and zinc, produces onlya small restorative effect. Thus, there is a need for compositions andmethods of restoring or stabilizing photoreceptor function andameliorating the effects of deficient levels of endogenous retinoids.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of restoring or stabilizingphotoreceptor function in a vertebrate visual system. Syntheticretinoids can be administered to human or non-human vertebrate subjectsto restore or stabilize photoreceptor function, and/or to ameliorate theeffects of a deficiency in retinoid levels.

In one aspect, methods are provided for restoring photoreceptor functionin a vertebrate eye. The method generally includes administering to avertebrate having an endogenous deficiency in the eye an effectiveamount of a synthetic retinoid in a pharmaceutically acceptable vehicle.The synthetic retinoid binds to opsin in the vertebrate eye and forms afunctional opsin/synthetic retinoid complex. The synthetic retinoid canbe, for example, a synthetic retinoid of formula I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII or XIII. In certain embodiments, the syntheticretinoid is 9-cis-retinal. The synthetic retinoid can be locallyadministered to the eye such as, for example, by eye drops, intraocularinjection or periocular injection. The synthetic retinoid also can beorally administered to the vertebrate.

In another aspect, a method is provided for sparing the requirement forendogenous retinoid in a vertebrate eye. The method generally includesadministering to the eye a synthetic retinoid in a pharmaceuticallyacceptable vehicle, wherein the synthetic retinoid binds to opsin in thevertebrate eye and forms a functional opsin/synthetic retinoid complex.The synthetic retinoid can be, for example, a synthetic retinoid offormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII. Incertain embodiments, the synthetic retinoid is 9-cis-retinal. Theendogenous retinoid that is deficient can be, for example,11-cis-retinal.

In yet another aspect, a method of ameliorating loss of photoreceptorfunction in a vertebrate eye is provided. The method generally includesprophylactically administering an effective amount of a syntheticretinoid in a pharmaceutically acceptable vehicle to the vertebrate eye.The synthetic binds to opsin protein to form a functionalopsin/synthetic retinoid complex. The synthetic retinoid can be, forexample, orally administered or locally administered. The syntheticretinoid can be, for example, a synthetic retinoid of formula I, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII. In certainembodiments, the synthetic retinoid is 9-cis-retinal.

In a further aspect, a method of selecting a treatment for a subjecthaving or at risk for developing a diminished visual capacity isprovided. The method generally includes determining whether the subjecthas a deficient endogenous retinoid level, as compared with a standardsubject, and administering to the subject an effective amount of asynthetic retinoid in a pharmaceutically acceptable vehicle. Thesynthetic retinoid binds to opsin in the subject's eye. The subject canbe, for example, a human having Leber Congenital Amaurosis, RetinitisPunctata Albesciens, Congenital Stationary Night Blindness, FundusAlbipunctatus or Age-Related Macular Degeneration. In certainembodiments, the endogenous retinoid that is deficient is11-cis-retinal.

The synthetic retinoid can be, for example, orally or locallyadministered to a vertebrate, such as by local administration to thevertebrate eye. The synthetic retinoid can be, for example, a syntheticretinoid of formula I, III, IV, V, VI, VII, VIII, IX, X, XI, XII orXIII. In certain embodiments, the synthetic retinoid is 9-cis-retinal.

In yet further aspects, an ophthalmologic composition is provided thatincludes a synthetic retinoid in a pharmaceutically acceptable vehicle.The synthetic retinoid can be, for example, a synthetic retinoid offormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII. Incertain embodiments, the synthetic retinoid is 9-cis-retinal. Theophthalmologic composition can be, for example, eye drops, anintraocular injectable solution or a periocular injectable solution.

In a further related aspect, an oral dosage form is provided thatincludes an opsin-binding synthetic retinoid in a pharmaceuticallyacceptable vehicle. The synthetic retinoid can be, for example, asynthetic retinoid of formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, XII or XIII. In certain embodiments, the synthetic retinoid is9-cis-retinal.

In another aspect, a method is provided of treating Leber CongenitalAmaurosis in a vertebrate subject. The method generally includesadministering to the subject an effective amount of a synthetic retinoidin a pharmaceutically acceptable vehicle. The synthetic retinoid bindsto opsin in the vertebrate eye and forms a functional opsin/syntheticretinoid complex. The synthetic retinoid can be, for example, asynthetic retinoid of formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, XII or XIII. In other embodiments, the synthetic retinoid can be,for example, a synthetic retinoid of formula I, II, III, IV, V, VI, VII,VIII, IX, X, XI, XII or XIII, with the proviso that the syntheticretinoid is not 9-cis-retinal. In other embodiments, the syntheticretinoid is 9-cis-retinal.

The synthetic retinoid can be, for example, locally administered to theeye. In certain embodiments, the synthetic retinoid is locallyadministered by eye drops, intraocular injection, periocular injection,or the like. The synthetic retinoid also can be orally administered tothe subject.

In yet another aspect, a method is provided for treating RetinitisPunctata Albesciens, Congenital Stationary Night Blindness or FundusAlbipunctatus in a vertebrate subject. The method generally includesadministering to the subject an effective amount of a synthetic retinoidin a pharmaceutically acceptable vehicle. The synthetic retinoid bindsto opsin in the vertebrate eye and forms a functional opsin/syntheticretinoid complex. The synthetic retinoid can be, for example, asynthetic retinoid of formula I, II, III, IV, V, VI, VII, VIII, IX, X,XI, XII or XIII. In certain embodiments, the synthetic retinoid is9-cis-retinal.

The synthetic retinoid can be, for example, locally administered to theeye. The synthetic retinoid can be locally administered by, for example,eye drops, intraocular injection or periocular injection. The syntheticretinoid also can be orally administered to the subject.

In yet a further aspect, a method is provided for treating Age-RelatedMacular Degeneration in a vertebrate subject. The method generallyincludes administering to the subject an effective amount of a syntheticretinoid in a pharmaceutically acceptable vehicle. The syntheticretinoid binds to opsin in the vertebrate eye and forms anopsin/synthetic retinoid complex. For example, the synthetic retinoidcan bind to free opsin in the eye.

The synthetic retinoid can be, for example, a synthetic retinoid offormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII. Incertain embodiments, the synthetic retinoid is 9-cis-retinal. Thesynthetic retinoid can be, for example, locally administered to the eye.For example, the synthetic retinoid can be locally administered by eyedrops, intraocular injection or periocular injection. The syntheticretinoid also can be orally administered to the subject.

In another aspect, a method is provided of treating or preventing lossof night vision or contrast sensitivity in an aging vertebrate subject.The method generally includes administering to the subject an effectiveamount of a synthetic retinoid in a pharmaceutically acceptable vehicle.The synthetic retinoid can bind to opsin in the vertebrate eye and forman opsin/synthetic retinoid complex. For example, the synthetic retinoidcan bind to free opsin in the eye.

The synthetic retinoid can be, for example, a synthetic retinoid offormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII. Incertain embodiments, the synthetic retinoid is 9-cis-retinal. Thesynthetic retinoid can be, for example, locally administered to the eye.Suitable methods of local administration include, for example, by eyedrops, intraocular injection or periocular injection. The syntheticretinoid also can be orally administered to the subject. In certainembodiments, the synthetic retinoid is administered prophylactically tothe subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Changes in retinoid levels and interface between RPE and ROS inRpe65−/− mice gavaged with 9-cis-retinal. FIG. 1A, the levels ofall-trans-retinyl esters (closed circles) and 11-cis-retinal (closedsquares) in Rpe65+/+ compared with levels of all-trans-retinyl esters(open circles) in Rpe65−/− mice as a function of age. FIG. 1B, esteranalysis of 9-cis-retinal-treated and untreated Rpe65−/− mice. Rpe65−/−mice were treated with 25 μg of 9-cis-retinal starting at PND 7 everyother day until they were 1 month old. Note the y axis scale. FIG. 1C,age-related accumulation of all-trans-retinyl esters in Rpe65−/− mice(gray line with black data points) compared with the ester levels(circles) in animals treated with 9-cis-retinal starting at PND 7 (leftpanel) (25 μg every other day, and after PND 30 gavaged with9-cis-retinal (250 g) once a week) or PND 30 (right panel) gavaged with9-cis-retinal (250 μg) once a week. The levels of iso-rhodopsin intreated Rpe65−/− mice are indicated by triangles measured as11-cis-retinyl oximes. FIG. 1D, changes in the RPE-ROS interface inRpe65 mice treated with 9-cis-retinal. Rpe65−/− mice were treated with9-cis-retinal (200 μg each) at PND 7, 11, and 15 and analyzed when theywere PND 30 (panels c and d) and PND 90 (panels e and f). Rpe65−/− micewere treated with 9-cis-retinal (200 g each) at PND 30 and analyzed whenthey were PND 120 (panels g and h). Control retina from untreatedRpe65−/− mice at PND 7 and PND 30 is shown on the top (panels a and b,respectively). Only partially filled lipid-like droplet in early treatedmice (left column, arrow in panel c), and considerably improved RPE-ROSprocesses (right column) in all treated mice were observed. Scale bar, 1μm.

FIG. 2. Effects of light exposure on iso-rhodopsin levels in Rpe65−/−mice gavaged 9-cis-retinal and ERG responses after a long term treatmentwith 9-cis-retinal. FIG. 2A, comparison of iso-rhodopsin levels in1-month-old Rpe65−/− mice gavaged with a single dose of 9-cis-retinal(2.5 mg) and kept under 12 hours light/dark or at constant dark for 37days (n=4). FIG. 2B, the levels of rhodopsin or iso-rhodopsin in6-month-old Rpe65−/− mice. The rhodopsin levels in wild-type mice(column a) were compared with iso-rhodopsin in Rpe65−/− mice treatedtwice with 9-cis-retinal (2.5 mg each time) at 1 month old with 4-dayintervals (column c) and treated twice with 3-month (column d) or4-month (column e) intervals. No rhodopsin iso-rhodopsin was detected inuntreated Rpe65−/− mice (column b) (n=4). FIG. 2C, theintensity-dependent response of flicker ERGs in Rpe65+/+, Rpe65−/−,Rpe65−/− treated with 9-cis-retinal, and Rpe65−/− Rgr−/− mice. Theflicker recordings were obtained with a range of intensities of0.00040-41 cd·s/m² at a fixed frequency (10 Hz). Left panel, Rpe65+/+mice; right panel, Rpe65−/− with or without treatment (open and closedcircles, respectively) and Rpe65−/− Rgr−/− mice without treatment(closed triangles).

FIG. 3. Mean stimulus response curves (n=5) of Rpe65+/+ (squares) andRpe65−/− mice treated with 2.5 (filled circles), 1.25 (open circles),0.25 (filled triangles), and 0 (filled circles on same line as filledtriangles) mg of 9-cis-retinal. The differences in light sensitivitywere evaluated by comparing the half-saturating flash intensity (I0)obtained from fitting the mean data with an equation for exponentialsaturation.

R/R _(max)=1−exp^(ln2·ilo)  (Eq. 1)

where R is the peak amplitude of the response, Rmax is the amplitude ofthe maximum response, and i is the flash strength in photons/μm². Thesolid lines are the exponential saturation function fitted to data with10 (equivalent 500 nm photons/μm): 25 (Rpe65+/+), 164 (2.5), 1995(1.25), 3929 (0.25), and 3714 (0 mg of 9-cis-retinal). Inset, thekinetics of responses adapted by similar amounts (approximately 4-fold)by steady background illumination (336 equivalent 500-nm photons/μm²/s,black traces) in a Rpe65+/+ rod and by dark light (free opsin) in rodfrom Rpe65−/− mouse treated with 1.25 mg of 9-cis-retinal. Each trace isfrom a single rod and is the mean of 10-20 flashes either 6.25(wild-type) or 910 (Rpe65−/−1.25 mg of 9-cis-retinal (500 nmphoton/μm²/flash).

FIG. 4. Photosensitivity of 11-cis-7-ring-retinal isomers and substratespecificity of eye-specific RDHs. FIG. 4A, light sensitivity of11-cis-7-ring-retinals and 11-cis-7-ring-rhodopsin. The bleachingstudies were carried out as described under “Methods and Materials”(Example 2 (infra)). The conditions for oxime formation from each isomerare described below for FIG. 4C. Activities of 11-cis-RDH(detergent-purified human recombinant 11-cis-RDH-His6) and all-trans-RDH(prRDH expressed in Sf9 cells) were determined by monitoring theproduction of the corresponding [15-³H]retinol analog from the reductionof the 11-cis-ring-retinal isomer and pro-S-[4-³H]NADH (for 11-cis-RDH)or pro-S-[4-³H]NADPH (for prRDH) (31) as described under “Methods andMaterials” (Example 2 (infra)). The product was analyzed by normal phaseHPLC, collected, and quantified by scintillation counting. FIG. 4B, thepurification of 11-cis-ring-rhodopsin isomers was monitored by UVspectroscopy in each step. Trace a, the 71,700×g supernatant of11-cis-ring-rhodopsin isomer 3 (solubilized by 10 mMn-dodecyl-β-D-maltoside); trace b, the flow-through fraction after thesupernatant passed through a concanavalin A-Sepharose 4B column (see“Methods and Materials”, Example 2 (infra)); trace c, the fraction afterextensive wash of the concanavalin A-Sepharose 4B column; trace d, thepurified 11-cis-7-ring-rhodopsin isomer 3; and trace e, thephotobleached 11-cis-7-ring-rhodopsion isomer 3. FIG. 4C, normal phaseHPLC analysis of oxime derivatives of 11-cis-7-ring-retinal isomers 1-4in solution (HPLC traces i-viii, 1′ and 1″:11-cis-7-ring-retinal isomer1 oximes, syn and anti, respectively; 2′ and 2″:11-7-cis-ring-retinalisomer 2 oximes, syn and anti, respectively; 3′ and3″:1-cis-7-ring-retinal isomer 3 oxime, syn and anti, respectively; and4′ and 4″:11-cis-7-ring-retinal isomer 4 oximes, syn and anti,respectively) and in rhodopsin 3 (ix and x) without (i, iii, v, vii, andix) or with (ii, iv, vi, viii, and x) photobleaching. The11-cis-7-ring-rhodopsin was solubilized with n-dodecyl-β-D-maltoside andpurified over a concanavalin A-Sepharose 4B column. The purifiedfraction was subjected to photobleaching, and the chromophore(s) wasderivatized with hydroxylamine and analyzed by HPLC as described under“Methods and Materials” (Example 2 (infra)). As controls, isomers 1-4were also derivatized in the same elution buffer with or withoutphotobleaching. *, contains minor amounts of compound 2 because of theunresolved peaks between compounds 2 and 3; mAU, milliabsorption units.

FIG. 5. LRAT activity toward different retinoids. Time course of LRATactivity with four 11-cis-ring-7-ring-retinol isomers, an average of twoindependent studies. Below is LRAT activity with all-trans-retinol, thenative substrate for LRAT. Assays were performed as described under“Methods and Materials” (Example 2 (infra)).

FIG. 6. Dissociation of Gt in the presence of GTP as measured usinglight-scattering methods. FIG. 5A, the dissociation signal of the nativesample at pH 7.4 and 6.4 evoked by a dim flash (Rh*/Rh=2.4×10⁻⁴). Thesedata show that according to the well known pH/rate profile at pH 7.4, ahigher activity of receptor is observed compared with pH 6.4. FIGS. 5Band 5C, the dissociation signal of the photoproduct of Rh regeneratedwith the 11-cis-7-ring (isomer 1) (B) or 11-cis-6-ring (C) analogs at pH6.4 and 7.4, respectively, evoked by a bright flash (1350-fold intensityas compared with A). FIG. 5D, sensitivity of 11-cis-6-ring-Rh to NH₂OH(2.5 mM) at pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of restoring or stabilizingphotoreceptor function in a vertebrate visual system. Syntheticretinoids can be administered to restore or stabilize photoreceptorfunction, and/or to ameliorate the effects of a deficiency in retinoidlevels. Photoreceptor function can be restored or stabilized, forexample, by providing a synthetic retinoid act as an 11-cis-retinoidreplacement and/or an opsin agonist. The synthetic retinoid also canameliorate the effects of a retinoid deficiency on a vertebrate visualsystem. A synthetic retinoid can be administered prophylactically ortherapeutically to a vertebrate. Suitable vertebrates include, forexample, human and non-human vertebrates. Suitable non-human vertebratesinclude, for example, mammals, such as dogs, cats, horses and otherdomesticated animals.

The synthetic retinoids are retinals derived from 11-cis-retinal or9-cis-retinal, or are 9-cis-retinal. In certain embodiments, the“synthetic retinoid” is a “synthetic cis retinoid.” In otherembodiments, the synthetic retinoid is a derivative of 11-cis-retinal or9-cis-retinal, with the proviso that the synthetic retinoid is not9-cis-retinal. In yet other embodiments, the synthetic retinoid is notvitamin A. In some embodiments, a synthetic retinoid can, for example,be a retinoid replacement, supplementing the levels of endogenousretinoid. In additional embodiments, a synthetic retinoid can bind toopsin, and function as an opsin agonist. As used herein, the term“agonist” refers to a synthetic retinoid that binds to opsin andfacilitates the ability of an opsin/synthetic retinoid complex torespond to light. As an opsin agonist, a synthetic retinoid can sparethe requirement for endogenous retinoid. A synthetic retinoid also canrestore function (e.g., photoreception) to opsin by binding to the opsinand forming a functional opsin/synthetic retinoid complex, whereby theopsin/synthetic retinoid complex can respond to photons when part of arod or cone membrane.

Synthetic retinoids include 11-cis-retinal derivatives or 9-cis-retinalderivatives such as, for example, the following: acyclic retinals;retinals with modified polyene chain length, such as trienoic ortetraenoic retinals; retinals with substituted polyene chains, such asalkyl, halogen or heteratom-substituted polyene chains; retinals withmodified polyene chains, such as trans- or cis-locked polyene chains, orwith, for example, allene or alkyne modifications; and retinals withring modifications, such as heterocyclic, heteroaromatic or substitutedcycloalkane or cycloalkene rings.

In certain embodiments, the synthetic retinoid can be a retinal of thefollowing formula I:

R and R1 can be independently selected from linear, iso-, sec-, tert-and other branched alkyl groups as well as substituted alkyl groups,substituted branched alkyl, hydroxyl, hydroalkyl, amine, amide, or thelike. R and R1 can independently be lower alkyl, which means straight orbranched alkyl with 1-6 carbon atom(s) such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, or the like.Suitable substituted alkyls and substituted branch alkyls include, forexample, alkyls, branched alkyls and cyclo-alkyls substituted withoxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatom or othergroups. Suitable heteroatoms include, for example, sulfur, silicon, andfluoro- or bromo-substitutions.

In certain additional embodiments, R or R1 can be a cyclo-alkyl such as,for example, hexane, cyclohexene, benzene as well as substitutedcyclo-alkyl. Suitable substituted cyclo alkyl include, for example,cyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine,halogen, heteroatom or other groups. Suitable heteroatoms include, forexample, sulfur, silicon, and fluoro- or bromo-substitutions.

The synthetic retinoid also can be a derivative of an 11-cis-retinal or9-cis-retinal that has a modified polyene chain length of the followingformula II:

The polyene chain length can be extended by 1, 2, or 3 alkyl, alkene oralkylene groups. According to formula (II), each n and n₁ can beindependently selected from 1, 2, or 3 alkyl, alkene or alkylene groups,with the proviso that the sum of the n and n₁ is at least 1.

The synthetic retinoid also can be a derivative of an 11-cis-retinal or9-cis-retinal that has a substituted polyene chain of the followingformulas IIIa and IIIb:

Each of R1 to R9 can be independently selected from hydrogen, alkyl,branched alkyl, cyclo-alkyl, halogen, a heteroatom, or the like.Suitable alkyls include, for example, methyl, ethyl, propyl, substitutedalkyl (e.g., alkyl with hydroxyl, hydro alkyl, amine, amide) or thelike. Suitable branched alkyl can be, for example, isopropyl, isobutyl,substituted branched alkyl, or the like. Suitable cyclo-alkyls caninclude, for example, cyclohexane, cycloheptane, and other cyclicalkanes as well as substituted cyclic alkanes such as substitutedcyclohexane or substituted cycloheptane. Suitable halogens include, forexample, bromine, chlorine, fluorine, or the like. Suitable heteroatomsinclude, for example, sulfur, silicon, and fluoro- orbromo-substitutions. Suitable substituted alkyls, substituted branchalkyls and substituted cyclo-alkyls include, for example, alkyls,branched alkyls and cyclo-alkyls substituted with oxygen, hydroxyl,nitrogen, amide, amine, halogen, heteroatom or other groups. Inexemplary embodiments, the synthetic retinoid is 9-ethyl-11-cis-retinal,7-methyl-11-cis-retinal, 13-desmethyl-11-cis-retinal,11-cis-10-F-retinal, 11-cis-10-Cl-retinal, 11-cis-10-methyl-retinal,11-cis-10-ethyl-retinal, 9-cis-10-F-retinal, 9-cis-10-Cl-retinal,9-cis-10-methyl-retinal, 9-cis-10-ethyl-retinal, 11-cis-12-F-retinal,11-cis-12-Cl-retinal, 11-cis-12-methyl-retinal, 11-cis-10-ethyl-retinal,9-cis-12-F-retinal, 9-cis-12-Cl-retinal, 9-cis-12-methyl-retinal,11-cis-14-F-retinal, 11-cis-14-methyl-retinal, 11-cis-14-ethyl-retinal,9-cis-14-F-retinal, 9-cis-14-methyl-retinal, 9-cis-14-ethyl-retinal, orthe like.

The synthetic retinoid further can be derivative of an 11-cis-retinal or9-cis-retinal that has a modified ring structure. Suitable examplesinclude, for example, derivatives containing ring modifications,aromatic analogs and heteroaromatic analogs of the following formulaeIV, V and VI, respectively:

Each of R1 to R5 or R6, as applicable, can be independently selectedfrom hydrogen, alkyl, substituted alkyl, hydroxyl, hydroalkyl, amine,amide, halogen, a heteratom, or the like. Suitable alkyls include, forexample, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or the like.Suitable halogens include, for example, bromine, chlorine, fluorine, orthe like. Suitable heteroatoms include, for example, sulfur, silicon, ornitrogen. In formulae VI, X can be, for example, sulfur, silicon,nitrogen, fluoro- or bromo-substitutions.

The synthetic retinoid can further be a derivative of an 11-cis-retinalor 9-cis-retinal that has a modified polyene chain. Suitable derivativesinclude, for example, those with a trans/cis locked configuration,6s-locked analogs, as well as modified allene, alkene, alkyne oralkylene groups in the polyene chain. In one example, the derivative isan 11-cis-locked analog of the following formula VII:

R can be, for example, hydrogen, methyl or other lower alkane or branchalkane. n can be 0 to 4. m plus 1 equals 1, 2 or 3.

In a specific embodiment, the synthetic retinoid is a 11-cis-lockedanalog of the following formula VIII:

n can be 1 to 4.

In certain exemplary embodiments, the synthetic retinoid is9,11,13-tri-cis-7-ring retinal, 11,13-di-cis-7-ring retinal,11-cis-7-ring retinal or 9,11-di-cis-7-ring retinal.

In another example, the synthetic retinoid is a 6s-locked analog offormula IX. R1 and R2 can be independently selected from hydrogen,methyl and other lower alkyl and substituted lower alkyl. R3 can beindependently selected from an alkene group at either of the indicatedpositions.

In other embodiments, the synthetic retinoid can be a 9-cis-ring-fusedderivative, such as, for example, those shown in formulae X-XII.

In yet another embodiment, the synthetic retinoid is of the followingformula XIII.

Each of R1 to R15 can be independently selected from hydrogen, alkyl,branched alkyl, halogen, hydroxyl, hydroalkyl, amine, amide, aheteratom, or the like. Suitable alkyls include, for example, methyl,ethyl, propyl, substituted alkyl (e.g., alkyl with hydroxyl, hydroalkyl,amine, amide), or the like. Suitable branched alkyl can be, for example,isopropyl, isobutyl, substituted branched alkyl, or the like. Suitablehalogens include, for example, bromine, chlorine, fluorine, or the like.Suitable heteroatoms include, for example, sulfur, silicon, and fluoro-or bromo-substitutions. Suitable substituted alkyls and substitutedbranch alkyls include, for example, alkyls and branched alkylssubstituted with oxygen, hydroxyl, nitrogen, amide, amine, halogen,heteroatom or other groups. Each of n and n₁ can be independentlyselected from 1, 2, or 3 alkyl, alkene or alkylene groups, with theproviso that the sum of the n and n₁ is at least 1. In addition, R11-R12and/or R13-R14 can comprise an alkene group in the cyclic carbon ring.In certain embodiments, R5 and R7 together can form a cyclo-alkyl, suchas a five, six, seven or eight member cyclo-alkyl or substitutedcyclo-alkyl, such as, for example, those shown in formulae VII, VI, X,XI and XII.

In additional embodiments, the synthetic retinoid also can be9-cis-retinal. Alternatively, 11-cis-retinal can be used.

Methods of making synthetic retinoids are disclosed in, for example, thefollowing references: Anal. Biochem. 272:232-42 (1999); Angew. Chem.36:2089-93 (1997); Biochemistry 14:3933-41 (1975); Biochemistry21:384-93 (1982); Biochemistry 28:2732-39 (1989); Biochemistry 33:408-16(1994); Biochemistry 35:6257-62 (1996); Bioorganic Chemistry 27:372-82(1999); Biophys. Chem. 56:31-39 (1995); Biophys. J 56:1259-65 (1989);Biophys. J. 83:3460-69 (2002); Chemistry 7:4198-204 (2001); Chemistry(Europe) 5:1172-75 (1999); FEBS 158:1 (1983); J. American Chem. Soc.104:3214-16 (1982); J. Am. Chem. Soc. 108:6077-78 (1986); J. Am. Chem.Soc. 109:6163 (1987); J. Am. Chem. Soc. 112:7779-82 (1990); J. Am. Chem.Soc. 119:5758-59 (1997); J. Am. Chem. Soc. 121:5803-04 (1999); AmericanChem. Soc. 123:10024-29 (2001); J American Chem. Soc. 124:7294-302(2002); J. Biol. Chem. 276:26148-53 (2001); J. Biol. Chem. 277:42315-24(2004); J. Chem. Soc.—Perkin T. 1:1773-77 (1997); J. Chem. Soc.—PerkinT. 1:2430-39 (2001); J. Org. Chem. 49:649-52 (1984); J. Org. Chem.58:3533-37 (1993); J. Physical Chemistry B 102:2787-806 (1998); Lipids8:558-65; Photochem. Photobiol. 13:259-83 (1986); Photochem. Photobiol.44:803-07 (1986); Photochem. Photobiol. 54:969-76 (1991); Photochem.Photobiol. 60:64-68 (1994); Photochem. Photobiol. 65:1047-55 (1991);Photochem. Photobiol. 70:111-15 (2002); Photochem. Photobiol. 76:606-615(2002); Proc. Natl. Acad. Sci. USA 88:9412-16 (1991); Proc. Natl Acad.Sci. USA 90:4072-76 (1993); Proc. Natl. Acad. Sci. USA 94:13442-47(1997); and Proc. R. Soc. Lond. Series B, Biol. Sci. 233(1270): 55-761988) (the disclosures of which are incorporated by reference herein).

For an opsin protein, synthetic retinoids can be identified, forexample, by an expression system expressing the opsin protein. Suitableanimal models include, for example, RPE65−/− mice (see infra). Suitablenon-human animal models further include rat, mouse, primate systems.Such animal models can be prepared, for example, by promoting homologousrecombination between a nucleic acid encoding an opsin in its chromosomeand an exogenous nucleic acid encoding a mutant opsin. In one aspect,homologous recombination is carried out by transforming embryo-derivedstem (ES) cells with a vector containing an opsin gene, such thathomologous recombination occurs, followed by injecting the ES cells intoa blastocyst, and implanting the blastocyst into a foster mother,followed by the birth of the chimeric animal (see, e.g., Capecchi,Science 244:1288-92 (1989)). The chimeric animal can be bred to produceadditional transgenic animals.

Suitable expression systems can include, for example, in vitro or invivo systems. Suitable in vitro systems include for example, coupledtranscription-translation systems. Suitable in vivo systems include, forexample, cells expressing an opsin protein. For example, cells of avertebrate visual system can be adapted for culture in vitro, orrecombinant cell lines expressing an opsin protein can be used. The celllines are typically stable cell lines expressing the opsin protein.Synthetic retinoid can be added to the cell culture media, and the cellscultured for a suitable period of time to allow the production ofopsin/rhodopsin. Opsin and/or rhodopsin can be isolated (e.g., byimmunoaffinity). Isolated protein samples are examined to determine theamount of pigment formed, and absorbance maxima. Methods of introducingnucleic acids into vertebrate cells are disclosed in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y., 2001).

Recombinant cell lines expressing opsin protein can be prepared by, forexample, introducing an expression construct encoding an opsin proteininto a suitable cell line. The expression construct typically includes apromoter operably linked to a nucleic acid encoding an opsin protein,and optionally a termination signal(s). Nucleic acids encoding opsin canbe obtained, for example, by using information from a database (e.g., agenomic or cDNA library), by polymerase chain reaction, or the like. Forexample opsin encoding nucleic acids can be obtained by hybridization.(See generally Sambrook et al. (supra).) In a specific embodiment, anopsin encoding nucleic acid can be obtained by hybridization underconditions of low, medium or high stringency.

In certain embodiments, opsin encoding nucleic acids can be obtainedunder conditions of high stringency hybridization. By way of example,and not limitation, procedures using conditions of high stringency areas follows: Prehybridization of filters containing DNA is carried outfor 8 hours to overnight at 65° C. in buffer composed of 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48hours at 65° C. in prehybridization mixture containing 100 g/mldenatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 65° C. for 1 hour in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 minutes before autoradiography. Otherconditions of high stringency which can be used are well known in theart. (See generally Sambrook et al. (supra).)

The expression construct can optionally include one or more origins ofreplication and/or selectable marker(s) (e.g., an antibiotic resistancegene). Suitable selectable markers include, for example, thoseconferring resistance to ampicillin, tetracycline, neomycin, G418, andthe like. Suitable cell lines include, for example, HEK293 cells,T-REx™-293 cells, CHO cells and other cells or cell lines.

The UV-visible spectra of rhodopsin (comprising opsin and a syntheticretinoid) can be monitored to determine whether the synthetic retinoidhas formed a Schiff's base with the opsin protein. For example,acid-denatured, purified protein can be analyzed to determine whether anabsorbance maxima of approximately 440 nm is present, providing evidencethat the synthetic retinoid forms a Schiff's base with the opsinprotein. In additional embodiments, hydroxylamine treatment can be usedto confirm the Schiff's base is sequestered from the externalenvironment (infra).

Suitable synthetic retinoids also can be selected by molecular modelingof rhodopsin. The coordinates for rhodopsin crystal structure areavailable from the Protein Data Bank (1HZX) (Teller et al., Biochemistry40:7761-72 (2001)). In certain embodiments, the effects of amino acidsubstitutions on the structure of rhodopsin, and on the contacts betweenopsin and 1-cis-retinal, or a synthetic retinoid, can be determined bymolecular modeling.

In an exemplary embodiment, the coordinates for the rhodopsin crystalstructure from the Protein Data Bank (1 HZX) (Teller et al.,Biochemistry 40:7761-72 (2001)) are used to generate a computer model.The addition of hydrogen atoms and optimization can be done, forexample, using Insight II (InsightII release 2000, Accelrys, Inc., SanDiego, Calif.). Crystallographic water can be removed, and watermolecules introduced based on the accessible space in the extracellularregion. Typically, no minimization is performed before water is added. Awater layer (e.g., 5 Å thick) can be used to coat the extracellular partof rhodopsin as well as residues in contact with polar phospholipidsheads. All of the water molecules can be allowed to move freely, as isthe extracellular half of rhodopsin, with retinal. If no water cap isput on the cytoplasmic part of rhodopsin, this part of the molecule canbe frozen to prevent degradation of the model.

In certain embodiments, a water cap is put on the extracellular part ofrhodopsin (together with that part buried in membrane in contact withpolar heads of phospholipids). Water and the extracellular part ofrhodopsin can be allowed to move and the movement modeled at anysuitable frequency. For example, the movement of the modeled rhodopsincan be modeling at 100 ps simulations.

Synthetic retinoids can be contacted with an opsin protein underconditions suitable and for a period of time sufficient for theformation of an opsin protein/synthetic retinoid complex. The stabilityof the opsin/synthetic retinoid complex can be determined by methodsdescribed herein or as known to the skilled artisan. The opsin in theopsin/synthetic retinoid complex is stabilized when it exhibitsincreased stability (e.g., increased half-life when bound to thesynthetic retinoid as compared with free opsin (i.e., not bound toretinoid), is less sensitive to hydroxylamine, exhibits lessaccumulation in aggresomes, or the like).

The synthetic retinoid can be contacted with the opsin protein in vitroor in vivo. For example, the opsin protein can be synthesized in an invitro translation system (e.g., a wheat germ or reticulocyte lysateexpression system) and the synthetic retinoid added to the expressionsystem. In additional embodiments, the opsin protein can be contactedwith the opsin protein ex vivo, and then the complex can be administeredto a vertebrate eye.

A synthetic retinoid can be administered to vertebrate eyes having aretinoid deficiency (e.g., a deficiency of 11-cis-retinal), an excess offree opsin, an excess of retinoid waste products (see infra) orintermediates in the recycling of all-trans-retinal, or the like. Thevertebrate eye typically comprises a wild-type opsin protein. Methods ofdetermining endogenous retinoid levels in a vertebrate eye, and adeficiency of such retinoids, are disclosed in, for example, U.S.Provisional Patent Application No. 60/538,051 (filed Feb. 12, 2004) (thedisclosure of which is incorporated by reference herein). Other methodsof detennining endogenous retinoid levels in a vertebrate eye, and adeficiency of such retinoids, include for example, analysis by highpressure liquid chromatography (HPLC) of retinoids in a sample from asubject. For example, retinoid levels or a deficiency in such levels canbe determined from a blood sample from a subject.

In an exemplary embodiment, a blood sample can be obtained from asubject and retinoid types and levels in the sample can be separated andanalyzed by normal phase high pressure liquid chromatography (HPLC)(e.g., with a HP1100 HPLC and a Beckman, Ultrasphere-Si, 4.6 mm×250 mmcolumn using 10% ethyl acetate/90% hexane at a flow rate of 1.4ml/minute). The retinoids can be detected by, for example, detection at325 nm using a diode-array detector and HP Chemstation A.03.03 software.A deficiency in retinoids can be determined, for example, by comparisonof the profile of retinoids in the sample with a sample from a normalsubject.

As used herein, absent, deficient or depleted levels of endogenousretinoid, such as 11-cis-retinal, refer to levels of endogenous retinoidlower than those found in a healthy eye of a vertebrate of the samespecies. A synthetic retinoid can spare the requirement for endogenousretinoid.

As used herein, “prophylactic” and “prophylactically” refer to theadministration of a synthetic retinoid to prevent deterioration orfurther deterioration of the vertebrate visual system, as compared witha comparable vertebrate visual system not receiving the syntheticretinoid. The term “restore” refers to a long-term (e.g., as measured inweeks or months) improvement in photoreceptor function in a vertebratevisual system, as compared with a comparable vertebrate visual systemnot receiving the synthetic retinoid. The term “stabilize” refers tominimization of additional degradation in a vertebrate visual system, ascompared with a comparable vertebrate visual system not receiving thesynthetic retinoid.

In one aspect, the vertebrate eye is characterized as having LeberCongenital Amaurosis (“LCA”). This disease is a very rare childhoodcondition that effects children from birth or shortly there after. Itaffects both rods and cones in the eye. For example, certain mutationsin the genes encoding RP65 and LRAT proteins are involved in LCA.Mutations in both genes result in a person's inability to make11-cis-retinal in adequate quantities. Thus, 11-cis-retinal is eitherabsent or present in reduced quantities. In RP65-defective individuals,retinyl esters build up in the RPE. LRAT-defective individuals areunable to make esters and subsequently secrete any excess retinoids. ForLCA, a synthetic cis-retinoid can be used to replace the absent ordepleted 11-cis-retinal.

In another aspect, the vertebrate eye is characterized as havingRetinitis Punctata Albesciens. This disease is a form of RetinitisPigmentosa that exhibits a shortage of 11-cis-retinal in the rods. Asynthetic cis-retinoid can be used to replace the absent or depleted11-cis retinal.

In another aspect, the vertebrate eye is characterized as havingCongenital Stationary Night Blindness (“CSNB”) or Fundus Albipunctatus.This group of diseases is manifested by night blindness, but there isnot a progressive loss of vision as in the Retinitis Pigmentosa. Someforms of CSNB are due to a delay in the recycling of 11-cis-retinal.Fundus Albipunctatus until recently was thought to be a special case ofCSNB where the retinal appearance is abnormal with hundreds of smallwhite dots appearing in the retina. It has been shown recently that thisis also a progressive disease, although with a much slower progressionthan Retinitis Pigmentosa. It is caused by a gene defect that leads to adelay in the cycling of 11-cis-retinal. Thus, synthetic retinoids can beadministered to restore photoreceptor function by retinoid replacement.

In yet another aspect, the vertebrate eye is characterized as havingage-related macular degeneration (“AMD”). In various embodiments, AMDcan be wet or dry forms. In AMD, vision loss occurs when complicationslate in the disease either cause new blood vessels to grow under theretina or the retina atrophies. Without intending to be bound by anyparticular theory, excessive production of waste products from thephotoreceptors may overload the RPE. This is due to a shortfall of11-cis-retinal available to bind opsin. Free opsin is not a stablecompound and can spontaneously cause firing of the biochemical reactionsof the visual cascade without the addition of light.

Administration of a synthetic retinoid to the vertebrate eye can quenchthe deficiency of 11-cis-retinal and spontaneous misfiring of the opsin.In certain embodiments, administration of a synthetic retinoid canlessen the production of waste products and/or lessen drusen formation,and reduce or slow vision loss (e.g., choroidal neovascularizationand/or chorioretinal atrophy).

In yet other aspects, a synthetic retinoid is administered to an agingsubject. As used herein, an aging human subject is typically at least45, or at least 50, or at least 60, or at least 65 years old. Thesubject has an aging eye, which is characterized as having a decrease innight vision and/or contrast sensitivity. Excess unbound opsin randomlyexcites the visual transduction system. This creates noise in the systemand thus more light and more contrast are necessary to see well.Quenching these free opsin molecules with a synthetic retinoid willreduce spontaneous misfiring and increase the signal to noise ratio,thereby improving night vision and contrast sensitivity.

Synthetic retinoids can be administered to human or other non-humanvertebrates. Synthetic retinoids can be delivered to the eye by anysuitable means, including, for example, oral or local administration.Modes of local administration can include, for example, eye drops,intraocular injection or periocular injection. Periocular injectiontypically involves injection of the synthetic retinoid into theconjunctiva or to the tennon (the fibrous tissue overlying the eye).Intraocular injection typically involves injection of the syntheticretinoid into the vitreous. In certain embodiments, the administrationis non-invasive, such as by eye drops or oral dosage form.

Synthetic retinoids can be formulated for administration usingpharmaceutically acceptable vehicles as well as techniques routinelyused in the art. A vehicle is selected according to the solubility ofthe synthetic retinoid. Suitable ophthalmological compositions includethose that are administrable locally to the eye, such as by eye drops,injection or the like. In the case of eye drops, the formulation canalso optionally include, for example, isotonizing agents such as sodiumchloride, concentrated glycerin, and the like; buffering agents such assodium phosphate, sodium acetate, and the like; surfactants such aspolyoxyethylene sorbitan mono-oleate (also referred to as Polysorbate80), polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, andthe like; stabilization agents such as sodium citrate, sodium edentate,and the like; preservatives such as benzalkonium chloride, parabens, andthe like; and other ingredients. Preservatives can be employed, forexample, at a level of from about 0.001 to about 1.0% weight/volume. ThepH of the formulation is usually within the range acceptable toophthalmologic formulations, such as within the range of about pH 4 to8.

For injection, the synthetic retinoid can be provided in an injectiongrade saline solution, in the form of an injectable liposome solution,or the like. Intraocular and periocular injections are known to thoseskilled in the art and are described in numerous publications including,for example, Ophthalmic Surgery: Principles of Practice, Ed., G. L.Spaeth, W. B. Sanders Co., Philadelphia, Pa., U.S.A., pages 85-87(1990).

Suitable oral dosage forms include, for example, tablets, pills,sachets, or capsules of hard or soft gelatin, methylcellulose or ofanother suitable material easily dissolved in the digestive tract.Suitable nontoxic solid carriers can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like, (See, e.g., Remington “PharmaceuticalSciences”, 17 Ed., Gennaro (ed.), Mack Publishing Co., Easton, Pa.(1985).)

The doses of the synthetic retinoids can be suitably selected dependingon the clinical status, condition and age of the subject, dosage formand the like. In the case of eye drops, a synthetic retinoid can beadministered, for example, from about 0.01 mg, about 0.1 mg, or about 1mg, to about 25 mg, to about 50 mg, to about 90 mg per single dose. Eyedrops can be administered one or more times per day, as needed. In thecase of injections, suitable doses can be, for example, about 0.0001 mg,about 0.001 mg, about 0.01 mg, or about 0.1 mg to about 10 mg, to about25 mg, to about 50 mg, or to about 90 mg of the synthetic retinoid, oneto four times per week. In other embodiments, about 1.0 to about 30 mgof synthetic retinoid can be administered one to three times per week.

Oral doses can typically range from about 1.0 to about 1000 mg, one tofour times, or more, per day. An exemplary dosing range for oraladministration is from about 10 to about 250 mg one to three times perday.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

EXAMPLES Example 1

The visual process is initiated by the photoisomerization of11-cis-retinal to all-trans-retinal. For sustained vision, the11-cis-chromophore must be regenerated from all-trans-retinal. Thisrequires RPE65, a dominant retinal pigment epithelium protein.Disruption of the RPE65 gene results in massive accumulation ofall-trans-retinyl esters in the retinal pigment epithelium, lack of11-cis-retinal and therefore rhodopsin, and ultimately blindness. It waspreviously reported that in Rpe65−/− mice, oral administration of9-cis-retinal generated isorhodopsin (a rod photopigment) and restoredlight sensitivity to the electroretinogram (Van Hooser et al., Proc.Natl. Acad. Sci. USA 97:8623-28 (2000)). In this study, earlyintervention by 9-cis-retinal administration significantly attenuatedretinal ester accumulation and supported rod retinal function for morethan 6 months post-treatment. In single cell recordings, rod lightsensitivity was shown to be a function of the amount of regeneratedisorhodopsin; high doses restored rod responses with normal sensitivityand kinetics. Highly attenuated residual rod function was observed inuntreated Rpe65−/− mice. This rod function is likely a consequence oflow efficiency production of 11-cis-retinal by photo-conversion ofall-trans-retinal in the retina as demonstrated by retinoid analysis.These studies show that pharmacological intervention produces longlasting preservation of visual function in dark-reared Rpe65−/− mice andwill be a useful therapeutic strategy in recovering vision in humansdiagnosed with Leber congenital amaurosis caused by mutations in theRPE65 gene, an inherited group of early onset blinding and retinaldegenerations.

Introduction

Leber congenital amaurosis (LCA) is a group of conditions that causeblindness or severe visual impairment from birth. All show both rod andcone dysfunction, a negligible (not recordable) electroretinogram (ERG),and nystagmus. They result in early onset retinal dystrophy, which overtime may be accompanied by pigmentary changes in the retina, hence“amaurosis.” LCA is caused by defects in at least five different genesthat disrupt a variety of different cellular functions.

In approximately 12% of all LCA cases the gene for a 65-kDa protein(RPE65) of retinal pigment epithelium cells (RPE) is disabled. RPE65 isheavily expressed in RPE cells, where it plays an essential role in theretinoid cycle. This is a set of tightly interconnected events thatinvolve both photoreceptors and RPE cells. The photoisomerization of thevisual pigment chromophore (11-cis-retinal) produces all-trans-retinal,which is reduced in the photoreceptor, transferred to the RPE, convertedback to 11-cis-retinal, and then transferred back to the photoreceptorto regenerate the original visual pigment. The precise function of RPE65in retinoid processing is unknown.

Genetically engineered mice in which the gene for Rpe65 has beeneliminated (Rpe65−/−) exhibit changes in retinal morphology, function,and biochemistry that closely resemble the changes seen in human LCApatients. Both rod and cone function is severely disrupted, and the ERGis severely attenuated in Rpe65−/− mice. There is also a dramaticoveraccumulation of all-trans-retinyl esters in the RPE cells inlipid-like droplets and degeneration of the retina. Thus, the Rpe65−/−mouse provides the opportunity to gain insight into the cellular andmolecular origins and consequences of LCA as well as a means to testdifferent therapeutic strategies.

This study describes the results of an in-depth study of the changes inbiochemistry and function that occur in Rpe65−/− mice and show how theprogression of the disease can be interrupted and the functional effectsreversed by providing a supply of 9-cis-retinal. The goals were to: 1)examine the beneficial effects of 9-cis-retinal treatment on theprogression of the disease and on photoreceptor function; 2) evaluateusing single cell electrophysiology and ERG recording how 9-cis-retinaltreatment affected rod function and light-driven signals in the retina;and 3) investigate the biochemical basis for the low level of residualvision that persists in both LCA patients and Rpe65−/− mice.

Administration of 9-cis-retinal to Rpe65−/− mice produces and maintainsrod photopigment for more than 6 months in the dark. Early interventionwith 9-cis-retinal restores normal rod physiology and significantlyattenuates ester accumulation in the RPE, but only partially improvesretinal function as measured by ERG. These studies demonstrate thatpharmacological intervention produces long lasting preservation ofvisual function in dark-reared Rpe65−/− mice and is a useful therapy forrestoring vision in LCA patients.

Materials and Methods

Animals:

All of the animal studies employed procedures approved by the Universityof Washington Animal Care Committee and conformed with recommendationsof the American Veterinary Medical Association Panel on Euthanasia.Animals were maintained in complete darkness, and all of themanipulations were performed under dim red light employing all Kodak No.1 Safelight filter (transmittance, >560 nm). Typically, 2-3-month-oldmice were used in all of the studies. RPE65-deficient mice were obtainedfrom Dr. M. Redmond (NEI, National Institutes of Health) and genotypedas described previously (Redmond et al., Nat. Genet. 20:344-51 (1998);Redmond et al., Methods Enzymol. 316:705-24 (2000)). Retinal Gprotein-coupled receptor-deficient mice were generated and genotyped asdescribed previously (Chen et al., Nat. Genet. 28:256-60 (2001)). Doubleknockout Rpe65−/− Rgr−/− were generated by cross-breeding singleRpe65−/− and Rgr−/− mice to genetic homogeneity.

Analyses of Retinoids and Visual Pigments:

All of the procedures were performed under dim red light as describedpreviously (Van Hooser et al., Proc. Natl. Acad. Sci. USA 97:8623-28(2000); Jang et al., J. Biol. Chem. 276:32456-65 (2001); Palczewski etal., Biochemistry 38:12012-19 (1999)). In addition to previouslydescribed methods, retinoid analysis was performed on an HP 1100 serieshigh pressure liquid chromatograph (HPLC) equipped with a diode arraydetector and HP Chemstation A.07.01 software, allowing identification ofretinoid isomers according to their specific retention time andabsorption maxima. A normal phase column (Beclanan Ultrasphere Si 5μ,4.6×250 mm) and an isocratic solvent system of 0.5% ethyl acetate inhexane (v/v) for 15 minutes followed by 4% ethyl acetate in hexane for60 minutes at a flow rate of 1.4 ml/minute at 20° C. (total 75 min) withdetection at 325 nm allowed the separation of 11-cis-, 13-cis-, andall-trans-retinyl esters. In addition, all of the study proceduresrelated to the analysis of dissected mouse eyes, derivatization, andseparation of retinoids have been described previously in detail (VanHooser et al., Proc. Natl. Acad. Sci. USA 97:8623-28 (2000)). Rhodopsinand iso-rhodopsin measurements were performed as described previously(Palczewski et al., Biochemistry 38:12012-19 (1999)). Typically, twomouse eyes were used per assay, and the assays were repeated three tosix times. The data are presented with S.E.M.

Light and Electron Microscopy:

Eye cups were prepared by removing the anterior segment and vitreous.The eyes were collected on ice at PND 1-28 on a weekly basis. “Thin”sections (1.0 min) were stained with Richardson's blue solution (1%) andsubjected to light microscopy. “Ultrathin” sections (0.05 μm) werestained with uranyl acetate/lead citrate and subjected to electronmicroscopy.

Preparation of Mouse RPE Microsomes

Fresh mouse eyes were enucleated immediately after cervical dislocationor CO₂ asphyxiation. The anterior segment, vitreous, and retina werecarefully removed under a microdissecting scope. Typically, 30-40 eyeswere dissected for each preparation. RPE cells were separated by placing12 dissected eyecups in 400 μl of 10 mM MOPS, pH 7.0, containing 1 μMleupeptin and 1 mM dithiothreitol and vigorously shaken for 20 minutes.The eyecups were then gently brushed with a fine brush to furtherdislodge the RPE cells. The cell suspension was removed, another aliquotof 400 μl of MOPS buffer was added, and the eyecups were shaken againfor minutes. The cell suspensions were combined and subjected toglass-glass homogenization. The homogenate was centrifuged at 10,000×gfor 10 min, and then the supernatant was centrifuged at 275,000×g for 1hour. The pellet was then reconstituted in 200 μl of the MOPS buffer andresubjected to glass-glass homogenization. The total proteinconcentration (typically 0.5-1 mg/ml) was determined by the Bradfordmethod. (See, e.g., Bradford, Anal. Biochem. 72:248-54 (1976).)

Isomerization of All-trans-retinol to 11-cis-Retinol using Mouse RPEMicrosomes:

The assay used for determining isomerization to 11-cis-retinol wasreported previously (McBee et al., Biochemistry 39:11370-70 (2000)).Briefly, 20 μl of bovine serum albumin (final concentration, 1%), 125 μlof 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane, pH 7.5, 101 ofATP (1 mM final concentration), 25 μM apo-recombinant CRALBP, 40 μl ofRPE microsomes (typically 25-50 g of total protein), and 0.5 μl of 4 mMall-trans-retinol in dimethylformamide. The reactions were incubated for2 hours at 37° C. The reaction was quenched using 300 μl of MeOH, andthe retinoids were extracted with 200 μl of hexane. The mixture wasshaken vigorously for 2 minutes and then centrifuged at 14,000 rpm for 4minutes for phase separation. The upper organic layer was removed, and a100 μl aliquot was separated and analyzed using an HP 1100 HPLC (BeckmanUltrasphere Si, 4.6 mm×250 mm, 1.4 ml/minute flow rate using 10% ethylacetate in hexane) equipped with HP Chemstation software (versionA.07.01).

Preparation of pro-S-[4-³H]NADH and pro-S-[4-³H]NADPH

Syntheses of pro-S-[4-³H]NADH and pro-S-[4-³H]NADPH were carried outwith L-glutamic dehydrogenase, NAD(P), and L-[2,3-³H]glutamic acid(PerkinElmer Life Sciences), as described previously (Jang et al., J.Biol. Chem. 276:32456-65 (2001); Jang et al., J. Biol. Chem.275:28128-38 (2000)).

RDH Assays:

The assays were carried out by monitoring the production of[15-³H]retinol (reduction of retinal) using 11-cis-retinal andpro-S-[4-³H]NAD(P)H as dinucleotide substrates in the presence orabsence of NADH (see, e.g., McBee et al., Prog. Retin. Eye Res.20:469-529 (2001)).

Oral Gavage:

Oral gavage was carried out as described previously (Van Hooser et al.,Proc. Natl. Acad. Sci. USA 97:8623-28 (2000)).

Intravenous Administration of Retinoids:

The chemicals were purchased from Sigma/Aldrich unless otherwisespecified. Solution A contained 10 mg of 9-cis-retinal, 75 mg ofCremophor EL, 1 mg of α-tocopherol, and 0.6 mg of benzoic acid suspendedin 1 ml of lactated Ringer's solution (Baxter). The mixture was vortexedfor 10 minutes and centrifuged for 10 minutes at 20,000×g, and theconcentration of 9-cis-retinal (7.7 mM) was determinedspectrophotometrically. Solution B contained 13 mg of 9-cis-retinal, 50mg of Cremophor EL, 10 mg of dipalmitoylphosphatidyl choline, and 40 mgof 2-hydroxypropyl-3-cyclodextrin suspended in 1 ml of lactated Ringer'ssolution (Baxter). The mixture was vortexed for 10 minutes andcentrifuged for 10 minutes at 20,000×g, and the concentration of9-cis-retinal (10 mM) was determined spectrophotometrically. Solutions Aand B (typically, 100 μl) were delivered to the mouse lateral tail veinemploying a 1-ml syringe equipped with a 27-gauge needle and a restrainttube.

Single Cell Recordings:

Mice were dark-reared from birth and sacrificed via cervicaldislocation, and the eyes were removed. The retina was isolated andstored on ice for up to 12 hours in HEPES-buffered Ames' solution (10 mMHEPES, pH adjusted to 7.4 with NaOH). Isolated rods were obtained byshredding a small piece of retina (roughly 1 mm²) with fine needles in a160-μl drop of solution. The drop was then injected into a recordingchamber mounted on the stage of an inverted microscope (Nikon Eclipse)equipped with an infrared video viewing system and continuouslysuperfused at 2-3 ml/minute with bicarbonate-buffered Ames' solutionwarmed to 37° C. (pH 7.4 when equilibrated with 5% CO2, 95% O₂). Theentire dissection was carried out under infrared illumination using adissecting microscope equipped with infrared-visible image converters.

An isolated rod was drawn by suction into a heat-polished, silanizedborosilicate electrode with an opening 1.2-1.5 μm in diameter. Theelectrode was filled with HEPES-buffered Ames' solution. The electricalconnections to the bath and suction electrode were made by NaCl-filledagar bridges that contacted calomel half-cells. Bath voltage was held atground by an active clamp circuit (Baylor et al. J. Physiol. 354:203-23(1984)). Membrane current collected by the suction electrode wasamplified by an Axopatch 200A patch clamp amplifier (Axon Instruments,Foster City, Calif.), filtered at 30 Hz (3 dB point) with an 8-poleBessel low pass filter, and digitized at 1 kHz. Light fromlight-emitting diodes with peak outputs at 470, 570, and 640 nm werecombined using a trifurcated fiber optic and focused on the preparationusing a water immersion lens in place of the microscope condensor. Thelight stimulus was spatially uniform and illuminated a circular area0.57 mm in diameter centered on the recorded cell. Light intensitieswere measured at the preparation and converted to equivalent 500-nmphotons (max for rod sensitivity) using the absorption spectrum ofrhodopsin and the measured light-emitting diode spectrum.

Mouse Electroretinograms:

Mice were dark-reared from birth and anesthetized (ketaject/xylaject, 65mg/kg intraperitoneally), and the pupils were dilated with tropicamide(1%). A contact lens electrode was placed on the eye with a drop ofmethylcellulose and a ground electrode placed in the ear. ERGs wererecorded and analyzed with the universal testing and electrophysiologicsystem 3000 (UTAS E-3000) (LKC Technologies Inc., Gaithersburg, Md.).The mice were placed in a Ganzfield chamber, and flicker recordings wereobtained from one eye. Flicker stimuli had a range of intensities(0.00040-41 cd·s/m²) with a fixed frequency (10 Hz).

Immunocytochemistry:

Rpe65 mice were divided into five groups: Rpe65−/−, Rpe65−/− that weregavaged with 9-cis-retinal and kept in the dark; Rpe65−/− that weregavaged with 9-cis-retinal, exposed to a flash, and kept in the dark for15 min; Rpe65+/+ that were kept in the dark; and Rpe65+/+ that wereexposed to a flash and kept in the dark for 15 minutes. For the flashstudies, dark-adapted mice were subjected to a flash (Sunpak 433D, 1 ms)from a distance of 2 cm. The retinas were fixed in 4% paraformalydehydein 0.13 M sodium phosphate, pH 7.4, for 15 hours at 4° C., and thetissues were transferred to 5, 10, or 15% sucrose in 0.13 M sodiumphosphate, pH 7.4, for 30 minutes each time and stored overnight in 20%sucrose in the same buffer at 4° C. The tissue was then transferred tooptimal cutting temperature cryoembedding compound and sectioned at 10μm. The cryosections were incubated overnight at 4° C. in mousemonoclonal anti-phosphorylated Rh A11-82P antibody diluted 1:10. TritonX-100 (0.1%) was included in all phosphate-buffered saline solutions tofacilitate antibody penetration. The controls were processed by omittingprimary antibodies from the incubation buffer. After incubation inprimary antibodies, the sections were rinsed with phosphate-bufferedsaline and then incubated with indocarbocyanine (Cy3)-conjugated goatanti-mouse IgG (1:200). The sections were rinsed in phosphate-bufferedsaline mounted in 5% n-propylgallate in glycerol and coverslipped.

Results

Early Treatment with 9-cis-Retinal Eliminates Oil-like Structures inRpe65−/− Mice:

In addition to the loss of photoreceptors, a defective interface betweenROS and RPE, RPE cells of Rpe65−/− mice contained numerous lipid-likedroplets. In young animals, empty vacuoles were observed in fixedelectron microscopy sections of RPE from Rpe65−/− mice but not incontrols. With increasing age (>PND 21), they were filled with adiffractive material that was retained during electron microscopysection preparation. This observation correlates with the excessiveaccumulation of all-trans-retinyl esters in Rpe65−/− mice (FIG. 1A, opencircles). Retinyl esters also accumulated with age in Rpe65+/+ mice,albeit at lower levels than for Rpe65−/− mice. By PND 21, approximately800 pmol/eye of retinyl esters accumulated compared with approximately40 pmol/eye for Rpe65+/+. For Rpe65+/+ mice, rhodopsin levels initiallyexceeded the amount of retinyl esters several-fold.

When PND 7 mice were treated with a 0.25-mg dose of 9-cis-retinal (10mg/ml) every other day until they were 30 days old, a dramatic change inthe ester accumulation was observed (FIG. 1B). With increasing age andcontinued administration of I dose (1.25 mg) per week, the amounts ofall-trans-retinyl esters increased, similar to Rpe65+/+, but the overallamounts of esters were dramatically suppressed with concomitantformation of iso-rhodopsin (FIG. 1C, left panel). Once deposited, theaccumulated esters in the RPE were not removed if the treatment beganafter more than 1 month of age (FIG. 1C, right panel). When younganimals or young adults were treated with 9-cis-retinal, the interfacecontacts between the RPE and ROS were improved (FIG. 1D, panels d, f,and h), and the vacuoles appeared to be only partially filled (FIG. 1D,panels c and e) over several months of this study. These observationssuggest that formation of the regenerated pigment significantly sloweddown accumulation of esters, but did not promote the complete removal ofthe all-trans-retinyl esters that had been deposited in the eye.

Long Term Effect of 9-cis-Retinal Treatment:

Treatment of mice with 9-cis-retinal produced a long lasting increase inphotopigment levels and a decrease in accumulation of all-trans-retinylesters. Rpe65−/− mice (1-month-old) were treated once (2.5 mg) with9-cis-retinal and then kept under either a 12-hour light/dark cycle, orunder 24 hour dark for 37 days. No appreciable depletion of retinal wasobserved under either set of conditions (FIG. 2A). These results suggestthat a single dose of 9-cis-retinal sustains iso-rhodopsin in theseanimals under normal laboratory conditions.

In another set of studies, the level of rhodopsin or iso-rhodopsin wasmeasured in 6-month-old Rpe65−/− mice (FIG. 2B). In these animals, theiso-rhodopsin levels were comparable for three groups of Rpe65−/− mice:mice treated twice with 9-cis-retinal (2.5 mg/dose) at PND 30 and 34,mice treated twice at PND 30 and 120, and mice treated twice at PND 30and 150. The 50% decrease of iso-rhodopsin in Rpe65−/− (FIG. 2, Bcompared with A) matches a similar decrease in rhodopsin in Rpe65+/+ asa function of age. The ester levels were reduced by >50% (compared withuntreated animals) and were unaffected by the frequency and dose of9-cis-retinal. No rhodopsin or iso-rhodopsin was detected in untreateddark-adapted Rpe65−/− mice.

9-cis-Retinal, reduced to 9-cis-retinol, can be stored in the eye andliver in the form of 9-cis-retinyl ester. When needed 9-cis-retinolwould be liberated by a retinyl hydrolase. To determine how large thereservoir of 9-cis-retinoids is in the eye and liver, a group of micewere treated with 9-cis-retinal (2.5 mg) and after 48 hour exposed tomultiple flashes at 1-hour intervals that bleached approximately 30-35%of rhodopsin/flash. iso-rhodopsin and 9-cis-retinyl esters weresignificantly depleted after more than three intense flashes. Retinylesters from liver and RPE were completely depleted after five flashes at24-hour intervals. Continuous shedding and resynthesis ofrhodopsin-containing ROS discs does not affect the long termpreservation of the visual pigment. Therefore, it appears that9-cis-retinal is, in a large part, recycled from phagocytizediso-rhodopsin to newly produced opsin molecules over an extended periodof time.

Physiological effects of 9-cis-Retinal Treatment:

Treatment of Rpe65−/− mice with 9-cis-retinal also provided long termimprovement of retinal function. The long term physiological effect of9-cis-retinal treatment was determined from single flash responses ofdifferent intensities and flicker ERG measurements on Rpe65+/+ andRpe65−/− mice. Previous studies showed a partial recovery of the ERGsensitivity 48 hours after oral 9-cis-retinal administration. Thispartial recovery persisted for more than 12 weeks in Rpe65−/− micetreated once at PND 30.

The flicker ERG in Rpe65+/+ mice reached a peak amplitude of 254.9±41.5μV at a light level of 0.015 cd·s/m² and 95.1±8.9 μV at 7.5 cd·s/m²(FIG. 2C, left panel). These data resemble the rod and cone dominant ERGresponses, respectively. In Rpe65−/− mice without treatment, the flickerERG reached a significantly smaller peak amplitude, 76.0±12.0 μV, at alight level of 7.5 cd-s/m² (FIG. 2C, right panel). Eight weeks after asingle treatment with 2.5 mg of 9-cis-retinal, the flicker ERG reachedpeak amplitudes of 137.3-24.4 μV at 0.059 cd·s/m² and 40.0±7.1 μV at 13cd·s/m² (FIG. 2C, right panel). These peaks were smaller and occurred ata higher light level than in the Rpe65+/+ mice; however, the response oftreated Rpe65−/− mice was 2.1 logarithmic units more sensitive and hadlarger amplitude than that of untreated mice. Thus, administration of9-cis-retinal provided a long term, partial recovery of the ERG.

Treatment with 9-cis-Retinal Eliminated Constitutive OpsinPhosphorylation:

To gain additional insight into the enzymatic processes of Rpe65 mice,several direct measurements of relevant enzymatic activities werecarried out. It is generally accepted that opsin has some signalingcapability. Immunolabeling on retina sections from Rpe65 mice using amonoclonal antibody against phosphorylated opsin could provide a cleanevaluation of this activity, whereas it would be expected that9-cis-retinal treatment would inhibit this activity.

The retinas from Rpe65+/+ mice and Rpe65−/− mice were fixed in constantdarkness. The ROS in Rpe65+/+ mice showed no labeling, and the ROS fromuntreated Rpe65−/− mice were labeled by a monoclonal antibody againstphosphorylated opsin. This labeling was abolished for Rpe65−/− mice(gavaged once at PND 30 and analyzed 48 hours post-treatment) treatedwith 9-cis-retinal. This 9-cis-treatment reduced phosphorylation ofopsin to levels comparable with those in normal rods. ROS fixed indarkness at 15 minutes following a single flash showed immunolabeling inboth Rpe65+/+ and Rpe65−/− mice treated with 9-cis-retinal. These datasuggest that opsin is constitutively phosphorylated in Rpe65−/− mice.These studies indicated a specific deficit in conversion ofall-trans-retinol to 11-cis-retinol and constitutive opsinphosphorylation but not in oxidation of 11-cis-retinol to11-cis-retinal. Constitutive opsin phosphorylation could be an importantelement in the pathogenesis of LCA.

To directly measure the isomerase activity, RPE microsomes were isolatedfrom Rpe65 mice using a novel procedure. In control studies using RPEmicrosomes from Rpe65+/+ mice, 11-cis-retinol was produced fromexogenously added all-trans-retinol only in the presence of RPEmicrosomes and CRALBP. 11-cis-Retinol was absent when CRALBP wasomitted, as well as when RPE microsomes or CRALBP were denatured byheat. 11-cis-Retinol was not detected in RPE microsomes from Rpe65−/−mice.

Because 11-cis-retinol dehydrogenase (11-cis-RDH) was purified in acomplex with RPE65 protein, oxidation of 1-cis-retinol was investigatedin RPE microsomes from Rpe65 mice. Strong activity was detected inRpe65+/+ and Rpe65−/− mice using NADPH and NADH as a dinucleotidecofactor. To distinguish NADPH-dependent activity from NADH-dependentactivity, the test for dehydrogenase activity was carried out in thepresence of nonradioactive NADH and [³H]NADPH. In such conditions, onlyNADPH-dependent dehydrogenase activity can be readily detected. Thedifferences between Rpe65+/+ and Rpe65−/− were insignificant becausethis activity is much higher than required for normal flow of retinoidsas determined from 11-cis-Rdh/mice (Jang et al., J. Biol. Chem.276:32456-65 (2001). These data suggest that RPE microsomes fromRpe65−/− mice contain high NADPH-dependent and NADH-dependentdehydrogenase activities. In addition, no differences were seen inimmunolocalization of 11-cis-RDH in the RPE of Rpe65 mice.

Treatment with 9-cis-Retinal Restores Normal Rod Function:

Because the ERG primarily reflects bipolar responses, the inability of9-cis-retinal to provide complete recovery could be due to residualdeficits in the photoreceptors or problems in signal transfer from rodsto bipolar cells. To determine whether 9-cis-retinal treatment couldrestore normal photoreceptor function, suction electrodes were used torecord the responses of single rods from Rpe65+/+ mice and untreated andtreated Rpe65−/− mice (gavaged once at PND 30 and analyzed 48 hourspost-treatment).

Light-evoked changes in circulating dark current were recorded fromouter segments of single rods from Rpe65+/+ mice or Rpe65−/− micegavaged 0, 0.25, 1.25, or 2.5 mg of 9-cis-retinal (once a day for twoconsecutive days preceding the study). Retinoid analysis revealed that300±25 pmol of iso-rhodopsin/eye was formed with a 2.5 mg dose of9-cis-retinal, 109.8 pmol of iso-rhodopsin/eye with a 1.25-mg dose, and85.6-6.2 of pmol/eye with a 0.25-mg dose. The nonlinear relation betweenthe dose of 9-cis-retinal and the iso-rhodopsin concentration presumablyreflects accumulation in the liver and other tissues. All of the rodtypes listed supported light responses that increased with increasingflash strength to reach a maximum (saturating) amplitude when the lightwas bright enough to cause all of the cGMP channels to close and fullysuppress the light-sensitive dark current of the cell. The responsefamilies from rods of each type show that the amplitude of thesaturating response increases with increasing doses of 9-cis-retinal.The relationship between mean dark current for each group of rods andthe dose of 9-cis-retinal is plotted. Light-sensitive dark current inRpe65−/− rods that received no supplemental 9-cis-retinal was 2.1±0.3pA, not significantly different from Rpe65−/− rods that received 0.25 mgof 9-cis-retinal (3.6±0.9 pA). Rod dark current increased with largerdoses of chromophore, reaching a value that was essentially the same asRpe65+/+ when mice where given 2.5 mg of 9-cis-retinal.

Two other properties of the Rpe65−/− flash response varied with theamount of supplemental 9-cis-retinal, response kinetics and lightsensitivity. To illustrate the kinetic differences, the average dimflash response (in the cells linear range) was determined for each rodtype. The mean responses from the five different sets of rods werescaled to the same peak amplitude and compared. Responses recorded fromRpe65+/+ and Rpe65−/− rods from mice treated with 2.5 mg of9-cis-retinal have essentially the same kinetics. The linear rangeresponses are superimposed, showing that the dim flash responses of thetwo different rod types have the same time-to-peak and recovery times.Responses recorded from rods from Rpe65−/− mice gavaged with 1.25 or0.25 mg of 9-cis-retinal are also essentially the same, with similartime-to-peak and recovery times; both are substantially faster thanthose of Rpe65+/+. The dim flash kinetics of rod responses from Rpe65−/−mice that received no supplemental 9-cis-retinal were intermediate; theywere faster than Rpe65+/+ but slower than rods from mice treated with1.25 or 0.25 mg of 9-cis-retinal.

The differences in light sensitivity between Rpe65+/+ rods and rods fromRpe65−/− mice are shown in FIG. 3, which plots the stimulus responsecurves for each of the five study conditions (Rpe65+/+ and Rpe65−/− micegavaged with 2.5, 1.25, 0.25, or 0 mg of 9-cis-retinal). Thehalf-saturating flash intensity was lowest in Rpe65+/+ rods(approximately 30 photons/μm²) and increased by factors of 6, 66, and131 in rods from mice gavaged with 2.5, 1.25, and 0.25 mg of9-cis-retinal, respectively. The light sensitivity of rods from micethat did not receive 9-cis-retinal was the same as rods from mice thatreceived the lowest dose (0.25 mg).

In the Absence of 9-cis-Retinal Treatment, 11-cis-Retinal is Produced inRpe65−/− Mice by Photoisomerization:

Rpe65−/− mice that were never exposed to light have 11-cis-retinal(identified as oximes) below detection level in conventionalmicroanalysis of retinoids. However, these mice respond to intenseillumination in ERG studies and in single cell recordings. To identifywhether 11-cis-retinal is produced by exposure to bright light, four oreight eyes were used for retinoid analysis instead of two eyes. ForRpe65−/−, no significant amounts of 11-cis-retinal were detected fordark-adapted animals. When more eyes were used for analysis, less than0.2 pmol/eye of 1-cis-retinal oximes were detected in a typicalchromatogram. All-trans-retinal (4.2±1.1 pmol/eye, n=8) was present, andan intense flash converted this aldehyde to 2.1±0.6 pmol/eye of11-cis-retinal. The retinoids were identified by the retention time withauthentic standards, and their UV spectra were measured during thechromatography. Next, it was important to determine whetherphotoisomerization resulted from the action of the “photo-isomerase”retinal G protein-coupled receptor protein. Double knockout Rpe65−/−Rgr−/− mice were generated, and retinoid analyses were carried out. Asignificant reduction in free all-trans-retinal was observed (2.2±0.2pmol/eye), but light flash photo-converted a similar fraction(approximately 50%) to 11-cis-retinal. To identify where in the RPE orin the retina these retinals are present, retina and RPE were separatedand analyzed individually (note that eight eyes were used). The majorityof all-trans-retinal was observed in the retina, whereas 11-cis-retinalwas present mostly in the RPE. Bleaching converted all-trans-retinal to11-cis-retinal that also resided in the retina. Once 11-cis-retinal isformed, its level does not change after 15, 30, or 120 minutes in thedark.

The ERG analyses of Rpe65−/− Rgr−/− mice were not qualitativelydifferent from the responses obtained from Rpe65−/− mice (FIG. 2C, rightpanel). Together, these results indicate that there is a retinalphotoisomerization pathway that produces 11-cis-retinal and regeneratesrhodopsin in prior bleached animals.

Different Methods of 9-cis-Retinal Delivery:

An important point was to compare different ways to deliver9-cis-retinoids with a goal to not only regenerate iso-rhodopsin but toalso build up reservoirs of cis-retinoids. Two methods were tested:gavage (as described previously (Van Hooser et al., Proc. Natl. Acad.Sci. USA 97:8623-28 (2000)) and intravenous injections. Intravenousinjection is an efficient way of delivering retinoids, and there were nomajor differences between aldehyde and alcohol forms or their isomericcompositions (11-cis-versus 9-cis-) of cis-retinoids. Intravenousinjection of 9-cis-retinal produced iso-rhodopsin when delivered withand without cyclodextrins. Retinal was cleared out rapidly from theblood but could be stabilized in the circulation for a longer time inthe presence of cyclodextrins (t1/2=12 hours versus 23 hours). Theaddition of cyclodextrin, possibly by extending the time of circulation,also led to higher accumulation of 9-cis-retinyl esters in the liver orRPE. A rapid clearance of 9-cis-retinal from the bloodstream makes itnecessary to give multiple intravenous injections to fully regenerateiso-Rh. This is not the case with gavage, in which the presence ofretinal in the bloodstream lasts for greater than 48 hours. Together,gavage and intravenous injections were effective in producingiso-rhodopsin in Rpe65−/− mice. The advantages and disadvantages of bothmethods are described under “Discussion” (infra).

Discussion

The Role of RPE65 and LCA:

Although the sequence of events that lead to the diseased state inRpe65−/− mice, the animal model of LCA, has not been established, it islikely that the primary defect is an interruption of the retinoid cycle.This cycle is responsible for regenerating the visual pigment throughthe enzymatic conversion of all-trans-retinal to 11-cis-retinal in theRPE and its return to the photoreceptor cell. Disruption of the normalretinoid flow between the RPE and photoreceptor can explain theoveraccumulation of retinal esters in the RPE. Furthermore, the failureto regenerate rhodopsin can account for diminished rod and cone lightsensitivity. The absence of 11-cis-retinal also increases free opsin inthe photoreceptor. A high level of free opsin produces substantialactivation of the phototransduction cascade, mimicking the effects ofcontinuous light exposure. This ongoing activity may cause the reductionin the thickness of the ROS layer and photoreceptor degeneration,effects also produced in animals exposed to continuous light. Thissequence of events may be further aggravated by the phosphorylation offree opsin, which has been shown in other studies to lead to retinaldegeneration.

Early treatment of Rpe65−/− mice with 9-cis-retinal inhibited theaccumulation of all-trans-retinal, improved the attachment contactsbetween RPE processes and ROS, led to dephosphorylation of opsin, andprevented the further progression of retinal degeneration. Theseobservations suggest that ester accumulation in the RPE and the presenceof high levels of active opsin in the photoreceptor may be the principlecauses of retinal degeneration in the Rpe65−/− mouse.

Rescued Rod Function:

The light sensitivity of rods from Rpe65−/− mice was restored in adose-dependent manner by dietary supplemental 9-cis-retinal. The highestdose supported rod responses with normal sensitivity and kinetics.Treatment with lower doses of 9-cis-retinal gave rise to rod responsesthat were desensitized and had faster kinetics, closely resembling thechanges in sensitivity and kinetics that occur during steady backgroundillumination in wild-type rods. The changes in the light sensitivity ofrod responses recorded from mice treated with the lower amounts of9-cis-retinal could be accounted for by a combination of two factors.One source of desensitization was a decrease in the effective collectingarea of the rod because of a reduction in both the amount of visualpigment and its quantum efficiency; the quantum efficiency ofiso-rhodopsin is about one-third that of rhodopsin. The remainingreduction in sensitivity could be explained by steady activation of thetransduction cascade by free opsin, producing an effect equivalent tothat caused by steady background illumination in wild-type rods.

Rods from Rpe65−/− mice that were not treated with 9-cis-retinal alsogenerated light responses that were strongly desensitized. The presenceof residual rod responses in untreated Rpe65−/− mice is consistent withprevious reports of reduced but present light responses in children withLCA. These results indicate that under these conditions the generationof light responses by flashes of intense light is most likely due to theproduction of 11-cis-retinal from the photoconversion ofall-trans-retinal in the retina. It is open to speculation whetherall-trans-retinal is free or coupled (either covalently ornoncovalently) to opsin. The preassociation of the chromophore and opsinwould make the formation of the light-sensitive 11-cis-retinal complex(i.e., rhodopsin) fast enough for it to be subsequently photoisomerizedand transduction-triggered within the period of a brief (10 ms) flash oflight.

Phototransduction in Rods of Rpe65 Mice:

The shifts in light sensitivity rods from treated and untreated Rpe65mice can be attributed to a decrease in the effective collecting area ofthe rod acting either alone (2.5 mg of 9-cis-retinal) or in addition todesensitization by an “equivalent background” because of a low level ofsteady activation of the transduction cascade by free opsin.

The effective collecting area (ECA) depends on the geometric collectingarea of the rod (A), the quantum efficiency of the pigment (QE), and thepigment density (α).

ECA=AQE·(1−10^(α1))  (Eq. 2)

where 1 is the path length. The pigment regenerated using 9-cis-retinalis iso-rhodopsin, which has about one-third of the quantum efficiency ofRh (0.22 versus 0.67). The biochemical measurements indicate that inmice gavaged with 2.5 mg of 9-cis-retinal, all of the pigment isiso-rhodopsin (no free opsin±10%) and is about 57% of the amount ofrhodopsin in Rpe65+/+ rods (i.e., 300 pM iso-rhodopsin versus 525 pMrhodopsin). The decreases in quantum efficiency and axial pigmentdensity would be expected to cause approximately 5-fold decrease ineffective collecting area of rods from mice fed with 2.5 mg of9-cis-retinal. This is in agreement with the 6-fold increase inhalf-saturating flash strength in rods from Rpe65−/− mice gavaged with2.5 mg of 9-cis-retinal, compared with rods of Rpe65+/+. In rods frommice treated with 1.25 and 0.25 mg of 9-cis-retinal, the axial densitiesof iso-rhodopsin were 21 and 16%, respectively, of the amount ofrhodopsin in Rpe65+/+ rods. By the same reasoning as above, thesechanges would be expected to increase the half-saturating flash strengthby 14.5- and 19-fold compared with Rpe65+/+. This is not enough toaccount for the observed shifts in sensitivity; rods from mice gavagedwith 1.25 and 0.25 mg of 9-cis-retinal are further desensitized byfactors of 4.5- and 6.8-fold, respectively.

The additional desensitization could be attributed to an equivalentbackground that acts like “dark light” to cause steady activation of thecascade. In separate studies on Rpe65+/+ rods the change in flashsensitivity by background illumination was described by theWeber-Fechner relationship:

S/S ^(d) f=1/1+I _(b) /I _(b)  (Eq. 3)

where St is the flash sensitivity in steady light, S is flashsensitivity in darkness, I_(b) is the background light intensity, and Iis the background intensity (10⁸ photons/μm²/s) that reduces the flashsensitivity by half its dark value. Hence, background intensities of 378and 648 photons/m²/s would be expected to cause 4.5- and 7-fold changesin flash sensitivity. With an effective collecting area of 0.5 μm² andan integration time of 0.3 seconds, these background intensitiescorrespond to equivalent activation in Rpe65+/+ rods of 57 and 97 Rh*/s.

The equivalent background of residual free opsin was determined in thetreated Rpe65−/− rods by combining biochemical measurements of the freeopsin concentration with physiological estimates of desensitization. Thenumber of Rh molecules in a Rpe65+/+ rod is estimated to be about 2×10⁷(i.e., 3 mM rhodopsin in 0.02 μl). Biochemical measurements on rods fromRpe65−/− mice indicate that they make approximately 40% less pigmentthan Rpe65+/+48 hours after treatment. Thus, the number of iso-rhodopsinmolecules in rods from Rpe65−/− mice gavaged with 2.5 mg of9-cis-retinal would be about 1.2×10⁷. Smaller doses of 9-cis-retinal donot regenerate all of the available pigment to form iso-rhodopsin,causing there to be a pool of free opsin. The retinoid analysis suggeststhat the amount of free opsin in rods from Rpe65−/− mice gavaged with1.25 and 0.25 mg of 9-cis-retinal would be 63 and 72% of the totalamount of available pigment (i.e., 7.5-8.6×10⁶ molecules). For thisamount of free opsin to cause desensitization in the rods from Rpe65−/−mice that is equivalent to the desensitization in Rpe65+/+ rods causedby a steady light that bleaches 57 and 97 Rh*/s, about 1×10⁵ opsin wouldhave to activate the cascade as well as 1 Rh* (1.3-0.9×10⁵ opsin: Rh*).This value is broadly consistent with previous estimates of activationratio of free opsin:Rh* (i.e., 10⁶:1). The inset in FIG. 3 shows thatbackground light adaptation and adaptation by an equivalent (free opsin)background that desensitized the flash response by similar amounts hadsimilar effects on the kinetics of the dim flash response. This is alsoin general agreement with previous studies that showed the adaptationalchanges in the kinetics of the dim flash response were similar, whetheradaptation was due to background light or the equivalent backgroundassociated with dark adaptation.

The highly desensitized rod responses recorded from untreated Rpe65−/−mice did not show the acceleration in response kinetics seen in rodsfrom treated mice. There are several possible explanations for thisdifference. One possibility is that the activity of free opsin is lessin rods from untreated Rpe65−/− mice than in those from treated mice,perhaps because of phosphorylation of the opsin in untreated rods. Thisexplanation would require that treatment with a low dose of9-cis-retinal converts most or all of the remaining free opsin to astate of higher activity, perhaps through dephosphorylation. Anotherpossibility is that the activation and deactivation of the photopigmentare altered in the untreated mice. For example, it is not clear that thephotopigment created by photoconversion is identical to normalrhodopsin; for example, the opsin may still be phosphorylated.

The complete or nearly complete rescue of normal rod function aftertreatment with 9-cis-retinal contrasted with the partial rescue of thesensitivity of the electroretinogram. Because the electroretinogramprimarily reflects activity of bipolar cells, this difference indicatesthat responses in the rods are not properly transmitted across therod-bipolar synapse. It is possible this synapse does not developproperly in Rpe65−/− mice because of a lack of visual signals.Continuous treatment with 9-cis-retinal from birth may help remedy thisproblem.

Advantages and Disadvantages of 9-cis-Retinal Treatments:

Retinals can be delivered to the eye effectively by one (or acombination) of two methods: gavage and intravenous injection. The mosteffective delivery system is gavage, which restores visual pigment in1-2 days and also produces accumulation of 9-cis-retinyl esters in theliver and RPE microsomes. It is a highly reproducible procedure. Thereis a transient elevation of retinoids in the blood for 48 hours that isfollowed by recovery to the normal level. The only noticeable drawbackis that much of the retinoid is secreted rather than stored, requiring ahigher dose than other delivery methods.

Intravenous injection is also an effective method for retinoid deliveryto the eye, but it has the disadvantage of the retinoids being rapidlyeliminated from the bloodstream by the kidneys. This can be prevented tosome degree by “caging” retinal in a cyclodextrin net. For fullregeneration, multiple or large doses must be injected, causingpotential problems with local infection. To lower the amounts ofcirculating all-trans-retinoids, it would be helpful to inhibit livercarboxylesterase to prevent all-trans-retinal from being released to thebloodstream. Such inhibitors, if they are potent, are highly toxic,because they inhibit other processes that require hydrolase activity.General and mild inhibitors, such as vitamins K₁ and E, are effective tosome degree, but more specific inhibitors are needed to enhance thelevel of cis-retinoids in the bloodstream. Finally, intraocularinjection is an option in same cases.

There is not a large reservoir of cis-retinoids in the liver and RPE,most likely because of nonenzymatic conversion of free retinal orretinol to the all-trans isomer. However, the efficiency ofmanunalianvision is remarkable and worth consideration in light of cis-retinoidtherapy. For example, the mammalian retina contains approximately 10⁸photoreceptors. If each photoreceptor absorbs on average 1-2×10³photons/s, with a quantum yield of 0.65 (or 0.3 for 9-cis-retinal), thedaily requirement of 11-cis-retinal is only less than 1 μg, an amountthat could be easily delivered by dietary supplement even if themajority of retinoids are retained in liver or secreted. Therecommendations for vitamin A intake is 0.8 mg/day for men and 0.7mg/day for women, with the upper safety limit of 3 mg/day is only anestimate, because of lack of data.

Multiple gavages do not increase the amount of retinyl esters in theeye. In contrast, early intervention significantly lowers theaccumulation of all-trans-retinyl esters (FIG. 1). This could be one ofthe prerequisites of successful cis-retinoid therapy for retinaldiseases. The level of all-trans-retinyl esters in the RPE ispredetermined by the time of the intervention. If the treatment isinitiated very early in life, the esters only gradually increase withage, as in wild-type mice. The treatment does not remove the esters fromthe eye but prevents accumulation of the esters. One possibleexplanation is that the retina sends a signal that opsin is notregenerated, and this causes retinol capture from the blood circulationand retention as retinyl ester in RPE. When retinyl esters cannot beconverted to 11-cis-retinal, and the “opsin signal” is on, these twofactors ultimately lead to ester accumulation. The mechanism of suchcommunication is unknown on a molecular level.

In summary, this study provides evidence that administration of9-cis-retinal restores rod photopigment and rod retinal function formore than 6 months and that early intervention significantly attenuatesthe ester accumulation. Opsin in Rpe65−/− mice is constitutivelyphosphorylated in rods of Rpe65−/− mice, and this modification of thevisual pigment could be involved in the pathophysiology of LCA;fortunately, after 9-cis-retinal-treatment, opsin is dephosphorylated.Evidence is also provided that the source of 11-cis-retinal in Rpe65−/−mice results from photoisomerization of all-trans-retinal present in theretina and that other mechanisms in addition to photoisomerase retinal Gprotein-coupled receptor are involved in this process, as shown indouble Rpe65−/− Rgr−/− knockout mice. Electrophysiological data usingsingle cell recordings suggest that 11-cis-retinal is formed in situ inrod outer segments. These studies provide information about the etiologyof LCA on a molecular level and demonstrate that pharmacologicalintervention produces long lasting preservation of the visual functionin dark-reared Rpe65−/− mice.

Example 2

Phototransduction is initiated by the photoisomerization of rhodopsin(Rh) chromophore 11-cis-retinylidene to all-trans-retinylidene. Here,using rhodopsin regenerated with retinal analogs with different ringsizes, which prevent isomerization around the C11=C12 double bond, theactivation mechanism of this G-protein-coupled receptor wasinvestigated. 11-cis-7-ring-rhodopsin does not activate G-protein invivo and in vitro, and it does not isomerize along other double bonds,suggesting that it fits tightly into the binding site of opsin. Incontrast, bleaching 11-cis-6-ring-rhodopsin modestly activatesphototransduction in vivo and at low pH in vitro. These results revealthat partial activation is caused by isomerization along other doublebonds in more rigid 6-locked retinal isomers and protonation of keyresidues by lowering pH in 11-cis-6-ring-rhodopsins. Full activation isnot achieved, because isomerization does not induce a complete set ofconformational rearrangements of rhodopsin. These results with 6- and7-ring-constrained retinoids provide new insights into rhodopsinactivation and indicate a use of locked retinals, particularly11-cis-7-ring-retinal.

In vertebrate retinal photoreceptor cells, isomerization of the visualpigment chromophore, 11-cis-retinal to all-trans-retinal, triggers a setof reactions collectively termed the phototransduction cascade. Thephototransduction events are initiated by activated rhodopsin (Rh*) andprogress through a classical G-protein cascade, ultimately leading toneuronal signaling. Metarhodopsin II (or Meta II, Rh*), thecatalytically active intermediate generated by photoisomerization ofrhodopsin chromophore, contains all-trans-retinal covalently bound toLys296 of opsin via the deprotonated Schiff's base. Subsequently, MetaII undergoes reprotonation, and the photolyzed chromophore is hydrolyzedand released from opsin. The precise mechanism of rhodopsin activationby the photoisomerized chromophore is unknown.

The photobleaching process of rhodopsin has been investigated usingretinal analogs that contained an extra ring between C10 and C13, makingretinal non-isomerizable around the 11-cis double bond. An artificialvisual pigment with restricted C9-C11 motion forms normal photolysisintermediates, suggesting an importance of C11=C12 bond isomerization inthe activation of rhodopsin. More recently, it was reported that afterphotoisomerization, the β-ionone ring of the chromophore moves to a newposition during the transition to Meta II (Borhan et al., Science288:2209-12 (2000)). Jang et al. (J. Biol. Chem. 276:26148-53 (2001)showed using 6-ring-constrained retinal isomers and the crystalstructure of rhodopsin in the ground state that if this movement isrestricted, only residual activity could be observed. Locked retinalanalogs have also used to study visual transduction in vivo usingvitamin A-deprived rats. These animals had approximately half of thenormal complement of rhodopsin, and injection of locked retinal led tothe appearance of the analog pigment in the photoreceptors but withoutsignificant effect on the sensitivity of electroretinographic b-waveresponses recorded from rat eye. Interference from wild-type rhodopsinprevented full interpretation of the results.

The light-triggered events in photoreceptors are intimately intertwinedwith the regeneration reactions that involve a two-cell system,photoreceptor cells and the retinal pigment epithelial cells (RPE).Every photoisomerization caused by absorption of a photon iscounterbalanced by regeneration of rhodopsin with newly synthesized11-cis-retinal. The photoisomerized product all-trans-retinal releasedfrom Rh* is reduced to all-trans-retinol in photoreceptors and thenconverted back to 11-cis-retinal in the RPE in an enzymatic processreferred to as the visual cycle or the retinoid cycle (McBee et al.,Prog. Retin. Eye Res. 20:469-529 (2001). Several components of theretinoid cycle have been identified, although major enzymatic andchemical transformations still remain poorly understood.

One of the proteins involved in the retinoid cycle is RPE65, a highlyexpressed membrane-associated RPE protein with a molecular mass of 65kDa. This protein appears to form a complex with 11-cis-retinoldehydrogenase (11-cis-RDH). The function of RPE65 is unknown, but it isbelieved to be involved in retinoid processing. RPE microsomes washedwith high salt that removed greater than 95% RPE65 still retained mostof the isomerization activity. However, unexpectedly, Rpe65−/− mice hadan overaccumulation of all-trans-retinyl esters in the RPE in the formof lipid-like droplets. Further retinoid analysis revealed no detectable11-cis-products in either ester or alcohol forms. Electroretinogram(ERG) measurements of Rpe65−/− mice revealed that the rod and conefunctions were severely attenuated. Small amounts of 11-cis-retinal areproduced by photochemical reaction in situ in photoreceptor cells, andit was demonstrated that early intervention with cis-retinoids greatlyattenuates retinyl ester accumulation (see Example 1). This animal modelis very useful for studying in vivo properties of rhodopsin regeneratedwith synthetic retinal analogs that undergo photoactivation processesdifferently from 11-cis-retinal without interference from wild-typerhodopsin.

In this study, rhodopsin, regenerated with ring-constrained11-cis-retinal isomers and containing a 3-carbon bridge between C10 andC13 that prevents isomerization around the C11-C12 double bond, does notundergo significant isomerization and activation in vivo or in vitro. Incontrast, the bleaching of 11-cis-6-ring-rhodopsin (2-carbon bridge)leads to isomerization along other double bonds and produces activespecies of rhodopsin at low pH that trigger phototransduction events invivo and in vitro as demonstrated by FTIR spectroscopy. These resultsprovide new insights into rhodopsin activation and concurrently indicatethat 6- and 7-ring-constrained retinoids will be useful in retinoidtherapy for retinal pathologies.

Methods and Materials

Synthesis of 11-cis-7-Ring-retinals:

11-cis-7-ring-retinals were synthesized according to publishedprocedures (Alcita et al., J. Am. Chem. Soc. 102:6372-6376 (1980);Fujimoto et al., Chirality 14:340-46 (2002); Caldwell et al., J. Org.Chem. 58:3533-37 (1993)). (See FIG. 4A for the identification of isomers1-4, also referred to as compounds 1-4.)

Photoisomerization of Rhodopsin Regenerated with 11-cis-7-Ring-retinals:

Preparation of rod outer segment, opsin, rhodopsin regeneration withretinals, and purification of rhodopsin on a concanavalin A-Sepharose 4Bcolumn were conducted as described previously (Jang et al., J. Biol.Chem. 276:26148-53 (2001)).

Phosphorylation of Rhodopsin Regenerated with 11-cis-Retinal and11-cis-7-Ring-retinals:

Regenerated rhodopsin (2 mg/ml) was mixed in 100 μl of 100 mM sodiumphosphate buffer, pH 7.2, containing 5 mM MgCl₂, 0.5 mM [³²P]ATP(approximately 35,000 to approximately 50,000 cpm/nmol) and purifiedrhodopsin kinase (approximately 5 μg of protein), and the assay wascarried out as described previously (Palczewski et al., J. Biol. Chem.266:1294955 (1991)). Studies were performed in triplicate.

HPLC Activity Assay for RDH with 11-cis-7-Ring-retinal Isomers:

Activities of 11-cis-RDH (retinol dehydrogenase) and photoreceptorall-trans-retinal-specific RDH (prRDH) or all-trans-RDH were assayed bymonitoring the production of [15-³H]retinol isomers (reduction ofretinal isomers) (Jang et al, J. Biol. Chem. 275:21128-38 (2000)). Thereaction mixture (100 μl) contained MES (final concentration, 66 mM, pH5.5), 1 mM DTT, pro-S-[4-³H]NADH (16 μM) for purified 11-cis-RDH-His6(0.31 μg), (Jang et al., J. Biol. Chem. 275:21128-38 (2000)) orpro-S-[4-³H]NADPH (12 μM) for prRDH (expressed in Sf9 cells andsuspended in 20 mM BTP, 1 mM dithiothreitol, 1 μM leupeptin at a 1:49cell pellet/buffer ratio), and 2 μl of 11-cis-7-ring-retinal isomer (120μM) substrate stock added last to initiate the reactions. The reactionswere incubated at 33° C. for 10-20 minutes.

Lecithin:Retinol Acyltransferase (LRAT) Assay:

Fresh bovine eyes were obtained from Schenk Packing Co., Inc. (Stanwood,Wash.). Preparation of bovine RPE microsomes was described previously(Stecher et al., J. Biol. Chem. 274:8577-85 (1999)). The microsomes wereresuspended in 10 mM MOPS, 1 μM leupeptin, and 1 mM dithiothreitol to atotal protein concentration of approximately 5 mg/ml as determinedphotocolorimetrically (Bradford, Anal. Biochem. 72:248-54 (1976)).Aliquots were stored at 80° C. and were used within 1 month ofpreparation. To destroy endogenous retinoids, 2001 of aliquots of RPEmicrosomes were irradiated in a quartz cuvette for 5 minutes at 0° C.using a ChromatoUVE-transilluminator (model TM-15 from UVP Inc.). Allstudies were carried out under dim red light conditions.All-trans-retinol, 11-cis-retinol, and 11-cis-7-ring-retinols weredissolved in dimethylformamide to 1 mM concentration as determinedspectrophotometrically. To a 1.5-ml polypropylene tube containing 130 μlof 10 mM BTP, pH 7.4, 20 μl of 10% bovine serum albumin, and 10 μl of 10mM ATP (in 10 mM BTP (1,3-bis[tris(hydroxymethyl)-methylamino]propane),pH 7.4) was added 201 of UV-treated bovine RPE microsomes (approximately100 μg of total protein). 2 μl of 1 mM dimethylformamide solution of11-cis-7-ring-retinol then was added to the mixture and incubated at 37°C. for the indicated times. The reactions were quenched by the additionof 300 μl of MeOH and 3001 of hexane. Retinoids were extracted byvigorous shaking on a vortex for 5 minutes and then centrifuged at14,000 rpm for 4 minutes to separate hexane and aqueous layers. Thehexane extract (100 μl) was analyzed by a normal phase HPLC (4% ethylacetate/hexane). The studies were performed in duplicate, and the amountof retinoids was normalized.

LRAT Inhibition Assay:

The assay was performed as described above, but after preincubation with11-cis-7-ring-retinols for 15 minutes at 37° C., 2 μl of 1 mM solutionof all-trans-retinol or 11-cis-retinol was added, and reactions wereincubated for an additional minutes. For control, the reactions werepreincubated with 2 μl of dimethylformamide without11-cis-7-ring-retinols.

FTIR Spectroscopy:

Opsin membranes (24 M) (Sachs et al., J. Biol. Chem. 275:6189-94 (2000))were incubated overnight with 240 μM 11-cis-retinal or with the mixtureof either 11-cis-6-ring- or 11-cis-7-ring-retinal isomers in the BTPbuffer (20 mM BTP, pH 7.5, containing 1 mM MgCl₂ and 130 mM NaCl).Suspensions of membranes were centrifuged for 25 minutes at 100,000×gand resuspended in the BTP buffer to yield 2.2 mM rhodopsin (fromabsorption at 500 nm). For the measurements with the high affinityanalog of transducin (Gt)-(340-350), 8 mM VLEDLKSCGLF (SEQ ID NO:1) wasused. For H₂O/D₂O exchange, the pellet was resuspended three times indeuterated buffer. The buffer solution was removed, and the pellettransferred to a 30-mm-diameter temperature-controlled transmission cellwith two BaF₂ windows and a 3-μm polytetrafluoroethylen gasket. Thespectra were recorded using a Broker ifs 66-V spectrometer (Bartl etal., FEBS Lett. 473:259-64 (2000)). For all samples, Meta II minusrhodopsin difference spectra were produced.

Kinetic Light Scattering:

Light-scattering changes were measured as previously described (Heck etal., Methods Enzymol. 315:32947 (2000)). Measurements were performed in10-mm path cuvettes with 300-μl volumes in isotonic buffer (20 mM BTP,130 mM NaCl, and 1 mM MgCl₂, pH 6.4, as indicated in the legend to FIG.6) at 22° C. with a 5-ms dwell time of the A/D converter (Nicolet 400,Madison, Wis.). Samples contained membrane suspension (3 μM rhodopsin)reconstituted with purified Gt (0.8 M) and 1 mM GTP. Reactions weretriggered by flash photolysis of rhodopsin with a green (500-20 nm)flash attenuated by appropriate neutral density filters. The flashintensity was quantified photometrically by the amount of rhodopsinbleached and expressed in terms of the mole fraction of photoexcitedrhodopsin (Rh*/Rh).

Animals:

All animal studies employed procedures approved by the University ofWashington Animal Care Committee and conformed to recommendations of theAmerican Veterinary Medical Association Panel on Euthanasia. All animalswere maintained in complete darkness, and all manipulations were doneunder dim red light employing a Kodak No. 1 Safelight filter(transmittance>560 nm). Typically, 2-3-month-old mice were used in allstudies. RPE65-deficient mice were obtained from M. Redmond (NationalEye Institute, National Institutes of Health, Bethesda, Md.) andgenotyped as described previously (Redmond et al., Nat. Genet. 20:344-51(1998; Redmond et al., Methods Enzymol. 316:705-24 (2000)).

Analyses of Retinoids and Visual Pigments:

All procedures were performed under dim red light as describedpreviously (Van Hooser et al., Proc. Natl. Acad. Sci. USA 97:862328(2000); Jang. et al., J. Biol. Chem. 276:3245665 (2001); Palczewski etal., Biochemistry 38:12012-19 (1999)).

ERGs:

Mice were anesthetized by intraperitoneal injection with 15 μl/g bodyweight of 6 mg/ml ketamine and 0.44 mg/ml xylazine diluted with 10 mMphosphate buffer, pH 7.2, containing 100 mM NaCl. The pupils weredilated with 1% tropicamide. A contact lens electrode was placed on theeye with a drop of methylcellulose, and a ground electrode (a referenceelectrode) was placed in the ear. ERGs were recorded with the universaltesting and electrophysiologic system 3000 (UTAS E-3000) (LKCTechnologies, Inc.). The mice were placed in a Ganzfield chamber, andresponses to flash stimuli were obtained from both eyes simultaneously.Flash stimuli had a range of intensities (0.00020-41 candela s/m²), andwhite light flash duration was 10 ms. Two to four recordings were madewith >10-second intervals. Typically, 4-8 animals were used forrecording of each point in all conditions. All ERG measurements weredone within 10-40 minutes after anesthesia.

Immunocytochemistry:

The section preparation and immunolabeling using anti-phosphorylatedrhodopsin antibody, A1182P (a generous gift from P. Hargrave), werecarried out as described previously (Van Hooser et al., J. Biol. Chem.277:19173-82 (2002)).

Modeling:

Coordinates for bovine rhodopsin were taken from the Protein Data Bank(1 HZX). An addition of hydrogen atoms and all optimizations were donein Insight II (InsightII release 2000, Accelrys Inc, San Diego, Calif.)as described previously (Jang. et al., J. Biol. Chem., 276:26148-53(2001)).

Results

Synthesis of 1-cis-7-Ring-retinals and Modeling of the Active Site ofRhodopsin:

The total synthesis of 11-cis-locked retinal analog incorporating a7-membered ring was recently reported (Fujimoto et al., Chirality14:340-46 (2002)). This method was followed with modifications tosynthesize 11-cis-7-ring-retinals. The compound was prepared as amixture of four isomers. These isomers are well separated by normalphase HPLC and have UV-visible and ¹H NMR spectra identical to thosedescribed earlier under “Methods and Materials” (supra) (Caldwell etal., J. Org. Chem. 58:3533-37 (1993); Akito et al., J. Am. Chem. Soc.102:6370-72 (1980)). The conformation of isomer 3,11-cis-7-ring-retinal, overlaps to a high degree with 11-cis-retinal.All isomers fit into the binding site of rhodopsin as demonstrated bymolecular modeling using the x-ray structure of rhodopsin (Palczewski etal., Science 289:739-45 (2000); Teller et al., Biochemistry 40:7761-72(2001)) and energy minimization algorithms.

Susceptibility of 11-cis-7-Lock-Rhodpsin to Isomnerization, Reduction,and Esterification:

11-cis-7-Ring-retinals are more stable to thermal isomerization comparedwith 6-ring isomers. Bleaching these 7-ring-retinoids in solutionproduces a mixture of isomers with the least abundant isomer being9,11,13-tricis-retinal 1 (FIG. 4A), Rhodopsin regenerated with theseisomers was purified using concanavalin A column chromatography (FIG.4B). When bound to opsin, 11-cis-7-ring-retinal (isomer 3) appears to bemost stable to isomerization (FIG. 4C), whereas 11,13-dicis isomer 2converts readily to isomer 3 even in the dark, suggesting that opsinpromotes this isomerization. Overall, 7-ring-containing retinoids appearto be more stable in all conditions and undergo interconversion to alower degree compared with 6-ring-containing retinals. With theexception of the tri-cis isomer 1, they are poor substrates for1-cis-RDH. They are utilized by prRDH without discrimination amongdifferent isomers. These data again are different from 6-ring-retinoids.The activities of both dehydrogenases for the best substrates are onlyapproximately one-tenth of that of native retinals, suggesting thatthese substrates will be poorly utilized by dehydrogenases endogenous tothe visual system. These retinoids are also poor substrates for LRAT,and only a fraction can be esterified (FIG. 5). 11-cis-7-Ring-retinalsare ineffective inhibitors of LRAT when assayed in the presence ofall-trans-retinol or 11-cis-retinol as substrates.

Activity of 11-cis-7-Ring-Rhodopsin In Vitro:

To assess the light-induced transformation in rhodopsin regenerated with11-cis-7-ring-retinals, FTIR spectroscopy was employed to monitorspectral changes characteristic to different regions of opsin. Uponphotoactivation, rhodopsin regenerated with isomers 1 and 4 yielded onlyminor changes in the difference spectra compared with wild-typerhodopsin, whereas isomers 2 and 3 were inactive. These changes reflectisomerization around other double bonds, excluding the locked C11=C12double bond because the 11-cis-bond is locked. All of these spectra werepH-insensitive. FTIR reveals that the chromophore is changing itsgeometry upon bleaching, but the movements of the chromophore do notcause significant changes in hydrogen bonding or in protonation statesof carboxylic acids of rhodopsin. Consistent with the spectral data,light-scattering changes as a monitor of Gt activation yieldedexceedingly low but measurable pH-independent activity.

In Vivo Regeneration of Rhodopsin with 6- and 7-Ring Isomers:

To produce rhodopsin regenerated with retinoid analogs for in vivostudies, Rpe65 mice were generated by Redmond et al. (24). These miceare unable to produce substantial amounts of 11-cis-retinal (Van Hooseret al., Proc. Natl. Acad. Sci. USA 97:8623-28 (2000)). Rhodopsin inRpe65+/+ mice has a chromophore that is light-sensitive. However,rhodopsin regenerated with 11-cis-7-lock-retinals in vivo employingRpe65−/− mice produced light-insensitive rhodopsin that could bedetected in the difference spectra by the addition of 1% SDS. Theretinoid analysis revealed the presence of the expected amount of visualpigment. Although the injection of the mixture of 7-ring isomers yieldedonly one isomeric product (11-cis-7-ring-retinal, isomer 3), the mixtureof 6-ring-containing retinal produced three isomers as could bepredicted from previous work (Jang. et al., J. Biol. Chem. 276:26148-53(2001)). These results suggest that opsin is readily and preferentiallyregenerated in vivo with 11-cis-7-ring-retinal, which mimics thestructure of 11-cis-retinal.

Lack of Activity for 11-cis-7-Ring-Rhodopsin In Vivo:

To increase the sensitivity of the assay and to assess the properties ofrhodopsin regenerated with 7-lock isomers in vivo, Rpe65−/− mice weretreated with isomer 3. The activity (a- and b-wave) was unaffected bythe treatment as compared with samples with only Me₂SO. In contrast, ina positive control, 9-cis-retinal significantly increased sensitivity oftreated mice, even at low light intensities. Furthermore,11-cis-7-ring-rhodopsin was inactive in vitro in the phosphorylationassay using purified rhodopsin kinase.

In summary, these results demonstrate that 7-ring-retinal producesrhodopsin, which for the most part is inactive in all tested conditions.This finding was confirmed using complementary methods of differentsensitivities in vivo and in vitro such as FTIR, ERG, Gt activation, andphosphorylation as detection assays.

11-cis-6-Ring-Rh Is Active In Vivo and In Vitro:

Surprisingly, rhodopsin regenerated in vivo with 6-ring-containingretinal is active at higher bleaches. The a- and b-waves are clearlyelevated compared with the Me₂SO control. This result is consistent withthe minor activity of rhodopsin as previously measured (Bhattacharaya etal., J. Boil. Chem. 267:6763-69 (1992); Ridge et al., J. Biol. Chem.267:6770-75 (1992); Jang. et al., 276:26148-53 (2001)). The relativeactivity of rhodopsin and the pigments with locked analogs is asfollows. With membranes containing rhodopsin regenerated with 6-lockedanalogs, a 1350-fold intensity of the activating light flash is neededto evoke Gt activation rates comparable to wild-type rhodopsin.Consistent with the pH dependence observed in the FTIR spectra, theactivity is enhanced at acidic pH. This is in contrast to the well knownpH/rate profile of native rhodopsin (higher activity at pH 7.4 ascompared with pH 6.4). Besides the mechanistic implications of thisresult (see “Discussion,” infra), these data allow the exclusion of theidea that the activity of the locked analogs is merely because of traceamounts of endogenous 11-cis-retinal. Moreover, the activity ofrhodopsin regenerated with 6-locked analogs is sensitive tohydroxylamine, indicating a similar “open” conformation of thelight-activated photoproduct as compared with native Rh*. Consistentwith the findings in vivo, the activity of 11-cis-6-ring-rhodopsin ismarkedly higher than that of the 7-locked pigment.

The FTIR spectra indicate different protein-chromophore interactions ofthe ground state of 11-cis-6-ring-rhodopsin compared with the bleachedsample. At pH 7.5, the change in chromophore-protein interaction,indicated by a band at 1206 cm⁻¹, did not lead to significant changes inthe protein, and only residual activity could be detected (14). However,at pH 4.5, the same movements led to reorientation of hydrogen bonds andchanges in secondary structure, forming a Meta II-like product that isable to bind Gt-(340-350)-derived peptide. This finding suggests that pHinduces structural changes in opsin that render possible the interactionof the chromophore with the protein environment in the binding site. ThepK_(a) for this change is 5.4 and the Meta II-like structure decays witha half-width time comparable to Meta II regenerated with 11-cis-retinal.A band at 1713 cm⁻¹ in the Rho/Meta II difference spectrum assigned tothe protonation of Glu113 appears to be shifted to 1708 cm⁻¹ in the MetaII-like product regenerated with 11-cis-6-ring isomer at pH 4.5. Incontrast to the Meta II band at 1713 cm⁻¹, this band is not shiftedsignificantly when the sample is treated with D₂O, but the bond shapehas slightly changed. Interestingly, this band is still observed in theMeta II-like photoproduct of the E1113Q mutant of rhodopsin regeneratedwith 11-cis-6-ring isomer.

Discussion

The results of this study lead to conclusions on two different althoughrelated topics, namely the mechanism of activation of rhodopsin and theutilization of the retinoid analogs in vivo.

Rhodopsin Activation: New Lessons Learned from the Studies of RetinoidAnalogs:

This study revealed new important information on the activation process.Three sharply distinct classes of the chromophore-protein interactionwere found for 11-cis-7-ring- and 11-cis-6-ring-containing retinals and11-cis-retinal. 1) Rhodopsin regenerated with 11-cis-7-ring isomer hasonly 0.1% of wild-type activity; it is also inactive in both sensitiveERG and FTIR studies. This low activity could be a result of thepresence of a small amount of free opsin and consistent with theestimated activation ratio of free opsin:Rh*, i.e., 10⁶:1. Therefore, itappears that 11-cis-7-ring-retinal is stabilized by the opsin bindingpocket, forming stable 11-cis-7-ring-rhodopsin. The bleaching of9,11-dicis and 9,11,13-tricis results in a conversion to the most stableisomer, isomer 3, and FTIR spectra that are consistent with the lack ofGt activation. 2) In contrast, the possible movements of11-cis-6-ring-retinal along C9=C10 and C13=C14 double bonds aresufficient to overcome the trigger barrier in the activation ofrhodopsin. This activity can be clearly detected in vivo and in thelight-scattering assays, advancing previous measurements usingnucleotide uptake and phosphorylation assays. This active stateresembles Meta II in its sensitivity to hydroxylamine, in features ofthe FTIR spectrum, and in its interaction with Gt peptide. However, thebleaching of 11-cis-6-ring-Rh leads to a Gt activation that ispH-dependent, whereas Meta II has a broad high activity over a widerange of pH values. This result suggests that isomerization along C9-C10and C13-C14 causes sufficient relaxation of rhodopsin around thechromophore to allow activation as was observed for chromophore-freeopsin. However, the activation of the rhodopsin is not achieved withproper “forced” protonation of key residues. The full activation occursonly when all-trans-retinylidene assumes the most extended conformationand that the β-ionone ring of the chromophore can act on its proteinenvironment. Although the mechanism of activation is still unclear, itis suspected that a profound relationship with another surprisingobservation, namely that the FTIR spectrum indicated an active species,whereas a spectral motif indicating protonation of the counterion Glu113 and salt bridge breaking was altered in its spectral properties.

The most obvious explanation for the pH-dependent activity would be thatH+ adjusts the retinal binding site in a way that is partiallyphotoactivatable and resembles a state generated during photoactivationof wild-type rhodopsin. Another non-exclusive explanation is that therestricted photochemistry of the 6-ring-retinals is sufficient topartially remove their reverse agonist-like property in a way thateventually allows protonation of key residue(s) (including Glu134) andthe formation of the active conformation (similar to theopsin/opsin*equilibrium). 3) In native Meta II, the energy content ishigh enough to force the receptor into the active conformation, even atneutral and basic pH values. The apparent pK_(a) of the light-inducedactive species of 11-cis-6-ring-Rh is 5.4, only one pH unit lower andthus approximately 1.5 kcal off the pK_(a) of a free Glu residue,whereas the protonated species of Meta II has pK_(a) of 6.7. Energy isrequired to protonate a residue at a pH higher than its native pK_(a) ofthe observed active species in native and11-cis-6-ring-retinal-regenerated rhodopsin.

In conclusion, it appears that the isomerization of retinal in therhodopsin binding pocket of native or 6-locked retinals leads toconformational changes of the protein that allow coupling with Gt.Interestingly, this property is specific and even distinguishes betweenclosely similar 6-ring- and 7-ring-containing retinals. This differenceprobably resides in the conformnation of both retinals in the activesite, their rigid nature imposed by the ring, and in the susceptibilityof 6-ring-containing retinal to isomerization.

Use of 6- and 7-Ring-containing Analogs in Leber Congenital Amaurosis:

Mutations in the RPE65 gene have been identified in patients diagnosedwith Leber congenital amaurosis (LCA) (Leber, Arch. Ophthabnol. (Paris)15:1-25 (1869); Marlhens et al., Nat. Genet. 17:139-41 (1997)), anautosomal recessive childhood-onset severe retinal dystrophy (Gu et al.,Nat. Genet. 17:194-97 (1997)), and autosomal recessive retinitispigmentosa (Morimura et al., Proc. Natl. Acad. Sci. USA 95:3088-93(1998)). LCA is characterized by congenital blindness or by poor centralvision, slight fundus changes, nearly absent electroretinogram signal,nystagmus, reduced papillary reactions, occasional photophobia(Schappert-Kimmijser et al., Opthalmologica 137:420-22 (1949)), eventualpigmentary degeneration of the retina, the absence of rodphotoreceptors, remnants of cones, clumping of pigment in RPE, and anabsence of chorioretinal adhesions (Leber, Arch. Ophthalmol. (Paris)15:1-25 (1869); Kroll et al., Arch. Ophthalmol. 71:683-690 (1964)). Thegenetic abnormalities of LCA involve genes from different physiologicalpathways (Cremers et al., Hum. Mol. Genet. 11:1169-76 (2002)), and RPE65gene mutations account for approximately 12% of all LCA cases (Thompsonet al., Invest. Ophthal. Vis. Sci. 41:4293-99 (2000)).

Several therapeutic approaches to treating LCA have been proposed. Thesemethods include RPE transplantation, gene replacement therapy, andpharmacological intervention. To date, most experimental therapeuticalinterventions for inherited degenerations in animals are aimed to slowdown the progression of degeneration. Encouragingly, the block in theretinoid cycle caused by RPE65 gene mutations may be overcomepharmacologically by the oral addition of 9-cis-retinal, therebycreating iso-rhodopsin. Within 48 hours after cis-retinoidadministration, rod photopigment was formed and rod physiology wasimproved dramatically, thus demonstrating that pharmacologicalintervention has the potential to restore vision when RPE65 is notpresent. This long term study on the effectiveness of the 9-cis-retinalintervention on restoration of visual function further lends support tothis idea.

The previous examples are provided to illustrate but not to limit thescope of the claimed inventions. Other variants of the inventions willbe readily apparent to those of ordinary skill in the art andencompassed by the appended claims. All publications, patents, patentapplications and other references cited herein are hereby incorporatedby reference.

1-58. (canceled)
 59. A method for treatment of an endogenous11-cis-retinal deficiency in a human eye, the method comprising:administering to a human subject an effective amount of 9-cis-retinal,wherein the 9-cis retinal is administered by intraocular or periocularinjection.
 60. The method according to claim 59, wherein the9-cis-retinal is injected into a vitreous of the eye.
 61. The methodaccording to claim 59, wherein the endogenous retinoid deficiency isassociated with Age-Related Macular Degeneration, Leber CongenitalAmaurosis, Retinitis Punctata Albesciens, Congenital Stationary NightBlindness, Fundus Albipunctatus or Retinitis Pigmentosa.
 62. The methodaccording to claim 59, wherein the human subject is at least 45, or atleast 50, or at least 60, or at least 65 years old.
 63. The methodaccording to claim 59, wherein said administering reduces or slowsvision loss.
 64. The method according to claim 59, wherein saidadministering restores or stabilizes photoreceptor function.
 65. Themethod according to claim 59, wherein said administering reduces orslows loss of night vision or contrast sensitivity in an aging humansubject.
 66. The method according to claim 59, wherein the 9-cis retinalis administered at a dose of about 0.1 mg to about 90 mg.
 67. A methodof restoring photoreceptor function in a human eye, the methodcomprising: administering to a human subject an effective amount of9-cis-retinal, wherein the 9-cis retinal is administered by intraocularor periocular injection.
 68. The method according to claim 67, whereinthe 9-cis-retinal is injected into a vitreous of the eye.
 69. The methodaccording to claim 67, wherein the human subject is at least 45, or atleast 50, or at least 60, or at least 65 years old.
 70. The methodaccording to claim 67, wherein the method of restoring photoreceptorfunction reduces or slows vision loss.
 71. The method according to claim67, wherein the 9-cis retinal is administered at a dose of about 0.1 mgto about 90 mg.
 72. A method of administering a synthetic retinoid to ahuman subject, wherein 9-cis retinal is administered by intraocular orperiocular injection to a human eye, whereby the 9-cis retinal binds toopsin in the human eye and forms a functional opsin/9-cis-retinalcomplex.
 73. The method according to claim 72, wherein the 9-cis-retinalis injected into a vitreous of the eye.
 74. The method according toclaim 72, wherein the human subject is at least 45, or at least 50, orat least 60, or at least 65 years old.
 75. The method according to claim72, wherein said administering reduces or slows vision loss in the humansubject.
 76. The method according to claim 72, wherein saidadministering restores or stabilizes photoreceptor function in the humaneye.
 77. The method according to claim 72, wherein said administeringameliorates the effects of retinoid deficiency in the human subject. 78.The method according to claim 77, wherein the retinoid deficiency isassociated with Age-Related Macular Degeneration, Leber CongenitalAmaurosis, Retinitis Punctata Albesciens, Congenital Stationary NightBlindness, Fundus Albipunctatus or Retinitis Pigmentosa.
 79. The methodaccording to claim 72, wherein the 9-cis retinal is administered at adose of about 0.1 mg to about 90 mg.